Air Toxics Hot Spots Program: Toluene Reference Exposure Levels

TSD for Noncancer RELs OFFICE OF ENVIRONMENTAL HEALTH HAZARD ASSESSMENT
August 2020
Air Toxics Hot Spots Program
Toluene
Reference Exposure Levels
Technical Support Document for the
Derivation of Noncancer Reference
Exposure Levels
Appendix D1
August 2020
Air and Site Assessment and Climate Indicator Branch
Office of Environmental Health Hazard Assessment
Appendix D1 Toluene
California Environmental Protection Agency
TSD for Noncancer RELs August 2020
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Appendix D1 Toluene
Toluene
Reference Exposure Levels
Technical Support Document for the Derivation of
Noncancer Reference Exposure Levels
Appendix D1
Prepared by the
Office of Environmental Health Hazard Assessment

Lauren Zeise, Ph.D., Director
Author
Albert Wang, Ph.D.
Nygerma L. Dangleben, Ph.D.
Daryn E. Dodge, Ph.D.
Rona M. Silva, Ph.D.
Technical Reviewers
John D. Budroe, Ph.D.
David M. Siegel, Ph.D.
Melanie A. Marty, Ph.D.
Daryn E. Dodge, Ph.D.
August 2020
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List of Acronyms
ACGIH American Conference of GTP glutamyl transpeptidase
Governmental Industrial Hygienists HSDB Hazardous Substances Data Bank
AFHK human toxicokinetic variability IPL interpeak latency
adjustment factors
LH luteinizing hormone
AIC Akaike Information Criterion
LOAEL Lowest observed adverse effect
ANOVA analysis of variance level
API American Petroleum Institute LWAE lifetime weighted average exposure
ATSDR Agency for Toxic Substances and MMEF peak expiratory flow rate at 50% of
Disease Registry FVC
AUC area under the curve MRI magnetic resonance imaging
BAEP brainstem auditory evoked potential MRL Minimal risk level
BAER brainstem auditory evoked NES Neurobehavioral evaluation system
response
NHANES National Health and Nutrition
BAL bronchoalveolar lavage Examination Survey
BMC Benchmark concentration NOAEL No observed adverse effect level
BMC05 Benchmark concentration producing NTP National Toxicology Program
a 5% response rate
OECD Organization for Economic
BMCL05 the 95% lower confidence limit of Cooperation and Development
the dose producing a 5% response
PBPK Physiologically based
rate
pharmacokinetics
BMD Benchmark dose
PEFR peak expiratory flow rate
BMDL estimation of the BMD 95% lower
ppb parts per billion
bound confidence limit
ppm parts per million
CAR Conditioned Avoidance Response
REL Reference exposure level
CCI color confusion index
RfC Reference concentration
CNS central nervous system
SDT signal detection task
CTI California Toxics Inventory
SMCBs small- and medium-sized
CVD cardiovascular disease
commercial buildings
ELISA Enzyme-linked immunosorbent
TAC Toxic air contaminant
assay
TolU unmetabolized toluene in urine
FAS fetal alcohol syndrome
TOTCI total confusion index
FEF25-75% Forced respiratory flow (25-75% of
forced vital capacity) TSD Technical Support Document
FEV1 Forced expiratory volume in 1 TWA Time-weighted average
second UDP Uridine diphosphate glucose
FSH follicle stimulating hormone UF Uncertainty factor
FVC Forced vital capacity VEP visual evoked potential
GC gas chromatography VER visual evoked response
GPT glutamic-pyruvic transaminase VOC Volatile organic compound
Toluene Reference Exposure Levels
(Methyl benzene; methyl benzol; phenyl methane; toluol)
CAS Registry Number 108-88-3
1. Summary
The Office of Environmental Health Hazard Assessment (OEHHA) is required to develop
guidelines for conducting health risk assessments under the Air Toxics Hot Spots Program
(Health and Safety Code Section 44360(b) (2)). In response to this statutory requirement
OEHHA developed a Technical Support Document (TSD) that describes acute, 8-hour, and
chronic Reference Exposure Levels (RELs). The TSD was adopted in December 2008
(OEHHA, 2008) and presents methodology for deriving RELs, and in particular explicitly
includes consideration of possible differential effects on the health of infants, children, and other
sensitive subpopulations, in accordance with the mandate of the Children’s Environmental
Health Protection Act (Senate Bill 25, Escutia, Chapter 731, Statutes of 1999, Health and Safety
Code Sections 39669.5 et seq.).
Toluene was chosen for a re-evaluation of its previously established RELs because it is one of
the prioritized toxic air contaminants selected for focused literature review in the Prioritization of
Toxic Air Contaminants Under the Children's Environmental Health Protection Act document
(OEHHA, 2001; Table 1A). The noncancer REL TSD guidelines (OEHHA, 2008) have been
used to develop the RELs for toluene presented in this document; this document will be added
to Appendix D of the TSD.
Toluene is a solvent that has been shown to cause sensory irritation (i.e., eye and upper
respiratory irritation) and central nervous system depression in humans at high acute airborne
exposures. Prolonged or repeated exposures have been associated with neurophysiological
decrements and acquired color vision impairment (dyschromatopsia). The non-cancer adverse
health effects of toluene also include severe disabilities of children if the substance is abused by
deliberate inhalation during pregnancy for its narcotic effect. Infants and children may be more
susceptible to the effects of toluene because their nervous systems are still developing, and
because metabolism of toluene in the first years of life is slower compared to that of adults.
This review includes relevant material published through October 2018 and is a technical review
of those studies specifically applicable to developing non-cancer acute, 8-hour, and chronic
inhalation RELs for toluene.
Appendix D1 1 Toluene
Toluene Acute REL

Reference exposure level 5,000 µg/m3 (5 mg/m3; 1,300 ppb; 1.3 ppm)
Critical effect(s) Headache, dizziness, slight eye and nose irritation.
Hazard index target(s) Nervous system; eyes; respiratory system.
Toluene 8-hour REL

Reference exposure level 830 μg/m3 (0.83 mg/m3; 220 ppb; 0.22 ppm)
Critical effect(s) Acquired color vision impairment (dyschromatopsia)
Hazard index target(s) Eyes
Toluene Chronic REL

Reference exposure level 420 μg/m3 (0.42 mg/m3; 110 ppb; 0.11 ppm)
Critical effect(s) Acquired color vision impairment (dyschromatopsia)
Hazard index target(s) Eyes
2. Physical & Chemical Properties (HSDB (2006) except as noted)

Description Colorless liquid
Molecular formula C7H8
Molecular weight 92.14 g/mol
Density 0.8636 g/cm3
Boiling point 110.6 °C
Melting point -94.9 °C
Vapor pressure 28.4 mm Hg at 25°C
Odor threshold 11 mg/m3 (2.9 ppm) (Amoore and Hautala, 1983) sweet,
pungent, benzene-like odor
Solubility Soluble in most common organic solvents, considered
insoluble in water (0.0526 g/100ml at 25 °C).
Conversion factor 3.76 mg/m3 = 1 ppm at 25° C
3. Major Uses and Sources

Toluene occurs naturally as a component of crude oil and is produced in petroleum refining and
coke oven operations (HSDB, 2006). As a result, automobile emissions are the principal source
of toluene to the ambient air. Xylenes, ethylbenzene, and benzene are often found together as
airborne co-pollutants with toluene. Toluene has been used as a sentinel chemical for benzene
in the context of air and water sample monitoring. Toluene is used as a solvent in paints,
coatings, synthetic fragrances, adhesives, inks, and cleaning agents. It has also been applied in
the production of polymers used to make nylon, plastic soda bottles, and polyurethanes and for
pharmaceuticals, dyes, cosmetic nail products, and the synthesis of organic chemicals
(Cosmetic Ingredient Review Panel, 1987).
The highest concentrations of toluene usually occur in indoor air from the use of common
household products (paints, paint thinners, adhesives, synthetic fragrances and nail polish) and
cigarette smoke (Sack et al., 1992).
In 2005, the California statewide mean outdoor monitored concentration of toluene was
approximately 2.19 μg/m3 (0.58 ppb ) (CARB, 2017). Estimates for toluene emissions from the
Statewide 2010 California Toxics Inventory (CTI) were 9867 tons from point sources, 4119 tons
from area-wide sources, 10,784 tons from on-road mobile sources, 6088 tons from other mobile
sources, and 46 tons from natural sources (CARB, 2010). Among the U.S. general public, the
mean toluene blood concentration was 1.96 μg/m3 (0.52 ppb) in adults and 0.53 μg/m3 (0.14
ppb) in children (Ashley et al., 1994; Sexton et al., 2005).
Toluene may also be released to the ambient air during the production, use, and disposal of
industrial and consumer products that contain toluene. Levels of toluene measured in rural,
urban, and indoor air averaged 1.3, 10.81, and 31.5 μg/m3 (0.35, 2.88, and 8.4 ppb),
respectively (USEPA, 1988). A geometric mean concentration of 9.8 μg/m3 (2.6 ppb) (range:
2.6–16.9 μg/m3 , 0.7–4.5 ppb) toluene was recorded for 12 northern California office buildings in
an indoor air quality study (Daisey et al., 1994). A recent study by Wu et al. (2011) identified a
geometric mean concentration of 4.47 μg/m3 (1.19 ppb) (range 0.44–200 μg/m3, 0.12 – 53 ppb)
toluene in 37 small- and medium-sized commercial buildings (SMCBs) in California. The main
source of toluene in these buildings was from motor vehicle emissions. The highest
concentrations of toluene are often found in buildings immediately after construction or
renovation (Brown, 2002). Toluene concentrations measured in portable and main building
classrooms in California during school hours were found to range between 4.7 and 21.4 µg/m3
(1.25 – 5.68 ppb) (Shendell et al., 2004).
4. Metabolism
Studies in human subjects and laboratory animals indicate that toluene is readily absorbed from
the respiratory and gastrointestinal tracts, but is only slowly absorbed through skin when applied
topically (ACGIH, 2010). Thus, toluene was not assigned a skin notation by ACGIH for worker
incidental contact with the liquid. Toluene is rapidly taken up into the bloodstream (Carlsson
1982) and distributed to various brain regions (Gerasimov et al. 2002). The metabolism of
toluene is depicted in Figure 1. The initial step in toluene metabolism is transformation by
cytochrome P-450 (CYP) enzymes, which occurs mainly in the liver. The most prominent of
these transformations is hydroxylation of the methyl group to form benzyl alcohol followed by
oxidation to benzoic acid (Tassaneeyakul et al., 1996; Nakajima et al., 1997). Most of the
benzoic acid is then conjugated with glycine to form hippuric acid, but a small portion can be
conjugated with UDP-glucuronate to form the acyl-glucuronide. A minor CYP-related pathway
involves a transient epoxidation of the aromatic ring to form either o- or p-cresol. The cresols
may undergo a variety of conjugation reactions, forming mainly sulfates and glucuronides.
Glutathione conjugation may also occur resulting in S-benzylglutathione and S-
benzylmercapturic acid (conjugation to benzyl alcohol), or S-p-toluyl glutathione and S-p-
toluylmercaptic acid (conjugation to the epoxidated ring). Much of the remaining toluene is
exhaled unchanged. The urinary excretion of toluene and its metabolites is rapid, with the major
portion excreted within 12 hours of exposure (Baelum et al., 1993).
Figure 1. Proposed pathways for toluene metabolism. Proposed enzymes are noted in
parentheses. CoA = coenzyme A; CYP = cytochrome P-450; DH = dehydrogenase; GSH =
glutathione; UDP = uridine 5'-diphosphate. Sources: US EPA (2005), Angerer et al. (1998),
IARC (1999), Nakajima and Wang (1994), Nakajima et al. (1997), and Tassaneeyakul et al.
(1996).
CYP2E1 is considered to be the most active hepatic isozyme in metabolizing toluene to benzyl
alcohol, particularly at low exposure concentrations (Nakajima and Wang, 1994; Nakajima et al.,
1997). Other isozymes, including CYP2B6, CYP2C8, CYP1A2, and CYP1A1 are also active in
forming benzyl alcohol. CYP1A2 is thought to be the main isozyme in forming o-cresol and other
minor metabolites. CYP2E1 is undetectable in the fetal liver, but develops rapidly within hours
after birth (Cresteil, 1998; Vieira et al., 1996). CYP1A2 and other hepatic CYPs begin to develop
by about one month of age. A majority of the CYPs, including CYP2E1, are near adult
concentrations by one year of age. Phase II enzymes, such as some glutathione-S-transferases
isozymes, are present in the fetal liver (Cresteil, 1998). Other isoforms that are present at very
low levels in the fetal liver increase soon after birth.
The abundance of CYPs in the human lung is only about 10% of the specific content found in
the liver (Zhang et al., 2006). However, the lung may represent as important a site for toluene
metabolism by the inhalation route. Many of the same isozymes involved in hepatic toluene
metabolism are also present in the lung, including CYP2E1, CYP1A1, CYP1A2, CYP2B6
(Bernauer et al., 2006; Hukkanen et al., 2002; Zhang et al., 2006). In addition, CYP2A13 is
expressed predominantly in the pulmonary tract and showed a high activity for benzyl alcohol
formation from toluene (Fukami et al., 2008). Similar to the liver, many of the pulmonary CYP
isozymes are not expressed or are expressed at very low levels at birth, but begin to develop
soon thereafter (Day et al., 2006; Fanucchi, 2006). The increase in CYP expression occurs in
parallel with the differentiation of the epithelium in growing children, which indicates that
enzymatic activity of many CYP isozymes are age-dependent. However, lung development
continues for approximately 8-12 years. Phase II enzymes appear to be expressed earlier in
development compared to CYP monooxygenases, suggesting a role in protecting the lung
during the period of rapid cellular differentiation and cell division (Fanucchi, 2006).
To conclude, the available evidence indicates that hepatic metabolism of toluene by infants may
not reach adult levels until about one year of age. However, metabolism of toluene by
pulmonary CYP isozymes in children may not reach adult levels until considerably later. Thus,
infants may be more susceptible to toxic effects of toluene due to slower metabolism during the
early years of life.
The toluene urinary metabolite hippuric acid is frequently used as a biological marker for
monitoring toluene exposure in the workplace (Morata et al., 1997; Thetkathuek et al. 2015).
However, it is a common urinary constituent from food sources, which may result in high
background levels of the metabolite in urine. Co-exposure to other compounds, including
styrene, ethyl benzene and benzoic acid, can result in interference since hippuric acid is also a
common metabolite of these compounds. Because the background level of hippuric acid will
mask occupational exposure at the TLV value of 20 ppm, the ACGIH (2010) has recommended
toluene in blood, unchanged toluene in urine, or the urinary metabolite o-cresol to be used as
markers of toluene exposure. O-cresol is a more sensitive indicator of exposure than hippuric
acid and can be used to assess low exposures to toluene.
With exposure to the same concentration of toluene, men were found to have a greater rate of
excretion (in µmol/min) of hippuric acid and o-cresol compared to women (Baelum et al., 1987).
The difference in excretion rate was correlated to body weight and to work level. However, when
corrected for urinary creatinine, the excretion rate of hippuric acid and o-cresol was greater in
women. The authors also noted there was a large inter-individual variation in urinary metabolite
levels among the test subjects. Under similar exposure conditions, the alveolar toluene
concentration tended to be higher in females than in males both at rest and during exercise
(Baelum et al., 1990). The excretion of the metabolites hippuric acid and o-cresol were found to
be correlated to the alveolar toluene concentration at rest but not during work. The tendency
towards a higher alveolar toluene concentration in females than in males could not be explained
by correlations to body size, nutritional status, or to lung volumes. The authors speculated that
this gender difference may be caused by the different fat distribution or by altered circulation.
Tardif et al. (1995) used a physiologically-based pharmacokinetic (PBPK) model, developed and
validated in the rat, to predict the uptake and disposition kinetics of a toluene and xylene mixture
in humans. They substituted the rat physiological parameters and the blood:air partition
coefficient with those of humans, and kept all other model parameters species-invariant. The
human toluene and xylene mixture PBPK model, developed based on the competitive metabolic
inhibition mechanism of interaction elucidated in the rat, simulated adequately the kinetics of
toluene and xylene during combined exposures in humans. The simulations with this PBPK
model indicate that an eight hour co-exposure to concentrations that remain below the current of
the American Conference of Governmental Industrial Hygienists Threshold Limit Value for
toluene (50 ppm or 190 mg/m3) and xylene (100 ppm) would not result in significant
pharmacokinetic interferences. This suggests that data from biological monitoring of worker
exposure to these solvents would be unaffected by co-exposures.
Pelekis et al. (2001) used validated PBPK models and simplified physiological-model-based
algebraic equations to translate ambient exposure concentration (1 ppm of a VOC for 30 days)
to tissue dose in adults and children for selected VOCs, including toluene. This approach
derived a pharmacokinetic (PK) Uncertainty Factor (UF) for human adult heterogeneity within a
human population and an adult-to-child PK UF based on the range of human physiological
parameters used in PBPK models. The adult-to-child UF assumed a child 10 kg in weight,
equivalent to an age between 1 and 2 years. The results indicated that there was no significant
difference between the UF for human adult PK variability and the PK variability for the adult-to-
child UF. Therefore, the authors concluded that the standard human adult PK default of 3.16 is
sufficient to protect children’s health as well. The lack of a significant difference between the two
PK UFs was primarily due to the hepatic clearance for highly metabolized VOCs, such as
toluene, being nearly identical for both adults and children. This model is supported by the
findings of Nong et al. (2006) below, which calculated an adult-child PK variability factor of 1.5
(i.e., the adult-child variability factor, calculated as the ratio of the 95th percentile value over the
50th percentile value for the adult and children 1-11 years of age).
The simulations run by Nong et al. (2006) indicated that neonates with higher levels of CYP2E1
(4.33 to 55.93 pmol/mg protein) as well as older children would have a lower AUC (0.16 to 0.43
μg/ml × hr). The latter values were closer to those simulated for adults. Similar results were
also obtained for 7 hr exposure to 17 ppm (64 mg/m3) toluene, a scenario previously evaluated
in human volunteers. The authors did not explicitly address whether CYP metabolism of toluene
is expected to be similar in adult men and women. The adult-child metabolism variability factor,
calculated as the ratio of the 95th percentile value for the following age groups over the 50th
percentile value for the adult, was 3.9 for neonate low metabolizers, 1.6 for infants (1 month–1
year), 1.5 for children (1–11 years), and 1.4 for adolescents (12–17 years). The lack of large
variability in the adult-child PK factor was explained by Nong et al. (2006) on the basis of
CYP2E1 levels in neonates, children and adults. CYP2E1 maturation occurs rapidly after birth,
and the enzyme content is a more sensitive parameter than hepatic blood flow rate in the
neonates whereas metabolism is more sensitive to blood flow in all other age groups. The
outcome is that hepatic metabolism of toluene would appear to be limited by enzyme content at
birth and evolve gradually to a flow-limited condition with increasing age. Not surprisingly, the
rate of metabolism and AUC of toluene in high metabolizing neonates and older children is
comparable to adults.
Mörk et al. (2014) derived human toxicokinetic variability adjustment factors (AFHK) for toluene
using previously validated PBPK models, based on toluene surrogate concentrations in blood
for six different age groups: 3 month-old and 1 year-old infants, 5, 10, and 15 year-old children,
and adults. The metabolism of toluene was modeled in both liver and lungs, was saturable, and
obeyed Michaelis-Menten kinetics. The resulting PBPK model was used to simulate the blood
toluene concentration profiles in each age group, assumed to be exposed to 1.3 ppm (4.9
mg/m3) toluene in the air through inhalation over 24 hours. The AFHK values were calculated as
the ratio between 95th percentile of the surrogate dose in each respective age group and the
median (50th) surrogate dose in the whole population. The ratios were 1.3 for 3 month-olds and
1.5 or 1.6 for all other age groups. In this model, only slight differences between age groups
were seen, similar to what was observed by Nong et al. (2006) above, except for < 1 month
neonates in Nong. The authors did not discuss what caused the difference in modeling results
between the two studies. However, Nong et al. used the median value for adults in the
denominator, rather than the population as a whole as Mörk et al. did. Gender differences in the
toluene AFHK were small. The AFHK for adult males and females were 1.5 and 1.6, respectively.
5. Acute Toxicity of Toluene

Acute Toxicity to Adult Humans
Acute toxic effects to the central nervous system (CNS), cardiovascular, hematopoietic,
reproductive, and respiratory systems, as well as to the liver, kidneys, skin, and sensory organs
have been reported for toluene (Fishbein, 1988). The CNS is a primary target organ for toluene
toxicity in both humans and animals for acute and chronic exposures. CNS dysfunction (which is
often reversible) and narcosis have been frequently observed in humans acutely exposed to low
or moderate levels of toluene by inhalation; symptoms include fatigue, sleepiness, headaches,
and nausea.
Toluene is one of the most widely abused chemical substances due to its sweet, pungent smell
and its narcotic effect, especially among teenagers and young adults because it is inexpensive
and easy to obtain (Lubman et al. 2008; Howard et al. 2011). Death has occurred at very high
levels of exposure from abuse of toluene-containing solvents (Paterson and Sarvesvaran, 1983;
Takeichi et al., 1986; Ameno et al., 1992; Shibata et al., 1994; Kamijo et al., 1998; Argo et al.,
2010; Tang et al., 2005). High concentrations of toluene have been found in the victims’ blood,
brain, lung, kidney, and liver. Autopsy findings include cerebral edema, congestion of cerebral
veins, pulmonary edema, and pancreatic and renal congestion. Acute abusers of toluene
solvent have also developed severe muscle paralysis, hypokalemia, renal tubular acidosis, and
hyperchloremic metabolic acidosis.
Case Reports
Two separate workplace incidents involving acute inhalation exposure to toluene in several
workers resulted in euphoria, drunkenness, dizziness, nausea, confusion, incoordination,
drowsiness, and loss of consciousness (Longley et al., 1967). The toluene concentrations were
estimated at 10,000 to 30,000 ppm (40,000 to 110,000 mg/m³) although no actual
measurements were made. No long-term follow-up of the exposed workers was conducted.
Cardiac arrhythmia has also been reported in humans acutely exposed to toluene (Dinwiddie,
1994). Following inhalation of a very high, but unknown level of toluene, an individual died from
severe CNS depression (Kamijo et al., 1998). Constriction and necrosis of myocardial fibers,
swollen liver, congestion and hemorrhage of the lungs, and tubular kidney necrosis were also
reported.
Kao et al. (2014) reported leukoencephalopathy (structure alteration of the brain white matter)
with magnetic resonance imaging (MRI) features atypical from those of chronic solvent
intoxication after a 49-year-old man exposed to probable very high levels of toluene during a
lacquer thinner explosion. The man ignited a fire while stripping a floor with lacquer thinner, and
was sent to the emergency department with second and third degree burns of the face and four
extremities, but he was alert and cooperative. In the following month of burn intensive care, in
spite of the aggressive treatments, the patient’s consciousness rapidly deteriorated to coma.
MRI of the brain at 35 days revealed multiple white matter lesions with perifocal edema. Brain
biopsy of the largest lesion at 3 months revealed diffuse white matter necrosis without evidence
of microorganism or significant inflammatory infiltration. The patient died of multiple organ failure
in the fourth month. Since the patient’s MRI features were different from those typical for chronic
(min. 3 years) paint thinner abusers, the authors hypothesized that the lacquer thinner explosion
exposed the patient to an extraordinarily high level of toluene, resulting in severe neurologic
deterioration, white matter necrosis and disruption of the blood-brain barrier. However, the
authors assumed toluene as the main component responsible for neurotoxicity without a blood
or urine sample.
Camara-Lemarroy et al. (2015) assessed 20 patients admitted to an emergency department due

to acute toluene intoxication by recent inhalation of toluene (paint thinner), among whom three
young females (average age 25.3 yrs) died of cardiac rhythm abnormalities, with altered mental
status, severe acidosis, hypokalemia and acute oliguric renal failure. In all the 20 patients, the
potential exposure levels were not estimated, while the most common symptoms were muscular
weakness or paralysis, altered mental status, nausea, vomiting and abdominal pain. Proteinuria
(renal glomerular injury), liver injury and rhabdomyolysis were also common. The authors
concluded that the hallmarks of acute toluene intoxication are hypokalemic paralysis and
metabolic acidosis.
Lin and Liu (2015) reported two cases of occupational toluene-poisoning and described their
brain MRI characteristics. Case 1 was a 31-year male factory worker who was admitted to the
emergency department (ED) due to refractory convulsions, lack of vitality, and general soreness
from painting work days and nights for one week in a room with no air conditioning. The
patient’s urine sample confirmed toluene intoxication, and cerebral MRI showed symmetric brain
lesions, common for neurological disorders. Case 2 was of a 61-year-old material-processing
factory worker who was admitted to the ED for deteriorating mental status and unsteady gait for
two months, due to prior painting work for long hours over 5 days in a poorly air-conditioned
room. The patient’s electroencephalography disclosed diffuse cortical dysfunction, and cerebral
MRI showed bilateral periventricular white matter change with corpus callosum involvement.
The patient’s urine sample confirmed the toluene intoxication diagnosis. In both cases,
increased signal intensity over the corpus callosum was observed in the initial brain MRI. This
finding was thought to be related to increased iron deposition and cytotoxic edema. The
abnormalities disappeared in the follow-up MRI scans. The authors suggested that the two
patients experienced acute or subacute brain injury due to toluene exposure, because with long
term toluene abusers, the cerebral atrophy is irreversible.
Djurendic-Brenesel et al. (2016) reported two cases of fatal intoxication with toluene due to glue
sniffing. Case 1 was of an 18-year-old male with a history of glue sniffing brought to an
emergency center for detoxification but who did not survive. Case 2 was of a 29-year-old male
found lying dead at a river bank. Using the gas chromatography/mass spectrometry (GC/MS)
method, the presence of toluene in biological samples were confirmed in both cases. Using the
GC/flame ionization detector (FID) method, the quantitative analysis of gastric content, femoral
blood, kidney, bile, liver and brain samples from both cases revealed toluene concentrations
ranging from 3.81 to 20.97 mg/kg.
Yasar et al. (2016) reported a 21-year-old man arrived at a military medical academy
emergency department with dyspnea and confusion. The authors reported the cause to be
intentional acute toluene inhalation. The authors could not obtain the history of toluene abuse,
frequency and duration of toluene exposure from the patient. The patient was diagnosed with
cardiomyopathy. After potassium replacement, and metoprolol and ramipril administration, the
patient had a dramatic clinical recovery and was discharged eight days after his hospitalization.
The authors stated that no relapse of cardiomyopathy was observed during the follow-up period
(timepoints unspecified).
Dharmarajan and Ammar (2017) reported a 31-year-old woman presented to the hospital with
generalized weakness and lower back and abdominal pain. Laboratory tests revealed
symptoms of toluene intoxication and elevated blood toluene level. The patient denied paint or
glue sniffing, but said that she had been exposed to paint while working on a friend’s house.
After potassium administration over 2 days, the symptoms resolved and the patient was
discharged.
Controlled Chamber Studies

Neurological effects (CNS, sensory irritation, neurobehavioral and psychometric
tests)
In a subacute chamber study, three volunteers were exposed to increasing concentrations of
toluene ranging from 50 to 800 ppm (190 – 3000 mg/m3) for up to 8 hours per day, 2 times per
week over 8 weeks (Von Oettingen et al., 1942). Only two exposures per week were conducted
to allow for sufficient recovery time between exposures. Concentrations of 50 and 100 ppm
(190 and 380 mg/m³) resulted in minor symptoms of fatigue, drowsiness, and headache toward
the end of the 8-hour exposures in some of the volunteers. At exposures of 200 to 400 ppm
(750 to 1,500 mg/m³), symptoms of muscular weakness, confusion, impaired coordination,
paresthesia (numbness of the skin), and nausea were also reported with some symptoms
lasting for several hours after exposure. Insomnia was also reported in all subjects. Exposure to
600 and 800 ppm (2300 and 3000 mg/m3) resulted in increased severity of symptoms with
considerable aftereffects in all 3 subjects (severe nervousness, muscular fatigue, and insomnia)
lasting up to several days. The authors concluded that 8-hour exposure to 200 ppm (750 mg/m3)
toluene produces definite impairment of coordination and reaction time.
Reaction time and perceptual speed were studied using psychophysiological tests in 12 young
male subjects exposed by inhalation to toluene concentrations of 100, 300, 500, and 700 ppm
(380, 1100, 1900, and 2600 mg/m3) in a successively increasing manner, each for a 20-minute
interval with a pause of 5 min between the second and third 20-min intervals (Gamberale and
Hultengren, 1972). Menthol was used to mask the odor of toluene. Statistically significant
impaired simple reaction time was apparent following exposure to 300 ppm (1100 mg/m3)
toluene. A statistically significant impairment in perceptual speed was observed at 700 ppm
(2600 mg/m3) toluene. No effects were observed at 100 ppm (380 mg/m3).
Stewart et al. (1975) exposed groups of male subjects in a chamber to a different concentration
of toluene (0, 20, 50 or 100 ppm, 0, 80, 190 or 380 mg/m3) each week over four-week period.
Three different groups of 2-4 subjects each, consisting of 1-, 3- and 7.5-hour exposure groups,
were exposed each day for up to 5 days per week. During the fifth week three groups of male
subjects (2-4 subjects per group), divided into 1-, 3- and 7.5-hour exposure groups, were
exposed to a fluctuating concentration of toluene between 50 and 150 ppm (190 and 570
mg/m3) each day for 5 days. In addition, three groups of female subjects (2 or 4 subjects per
group) were divided into 1-, 3- and 7.5-hour exposure groups and exposed to 100 ppm (380
mg/m3) toluene each day for 5 days.
All subjects immediately perceived a mild to strong odor for all toluene exposures when entering
the chamber, but it was usually not detectable after the first hour of exposure (Stewart et al.,
1975). Subjective responses included a 3-fold greater complaint of eye, nose and throat
irritation in the 3-hour male and female subjects (a total of 8 subjects) exposed to 100 ppm
toluene. However, no increase in sensory irritation was found in the 1- and 7.5-hour toluene-
exposed groups. The authors could not explain this discrepancy. The nostrils of both 7.5-hour
male subjects (n=2) exposed to a fluctuating concentration of toluene between 50 and 150 ppm
(190 and 570 mg/m3) were inflamed and infected on the fifth day of exposure, but only one
subject complained of sensory irritation. No increase in drowsiness, fatigue, sleepiness or
headache was observed among toluene-exposed groups.
Cognitive task testing revealed a decrement in the ability of 7.5-hour exposed females (n = 4) to
concentrate on the alertness test. However, no effect for this test was observed in 3-hour
exposed females or in exposed male groups. Other cognitive tests performed (time estimation,
coordination, arithmetic, and inspection tests) did not show an effect due to toluene exposure.
Spontaneous electroencephalograms and visual evoked responses (VER) were recorded in the
7.5-hour (n = 6) and fluctuating concentration subjects (n = 2). One of six male subjects
exposed to 100 ppm (380 mg/m3) toluene for 7.5 hours showed a significant increase in the
amplitude of the VER on the fifth day of exposure. A significant reduction in VER amplitude
during exposure to 150 ppm (570 mg/m3) toluene was also noted during week 5. The authors
suggest these responses are a sign of a pre-narcosis state. The authors concluded that there
was suggestive evidence of deleterious effects on subjects exposed to 100 ppm (380 mg/m3)
toluene.
Andersen et al. (1983) studied nasal mucus flow, lung function, psychometric performance, and
subjective responses in 16 young healthy males exposed to toluene concentrations of 10, 40,
and 100 ppm (38, 150, and 380 mg/m3) for 6 hours. The subjects were divided into four groups
and the exposures used a balanced Latin-square design with chamber exposures over a 4-day
period. No masking agents were used to disguise the odor of toluene. Data from the
psychometric performance tests were examined by analysis of variance with p < 0.05 used as
the level of significance. For statistical analysis of subjective tests, evaluations were collected
four times each day. The average score during exposure and the control score were ranked for
each subject, and nonparametric statistical methods (Friedman’s test and van Elteren’s test)
were applied to evaluate the effect of exposure.
A significant correlation of both increasing odor level and bad air quality was observed with
increasing toluene concentration. The odor impression was significantly different from control at
all toluene concentrations. Adaptation to the odor of toluene was noted during all exposures.
However, in three subjects the odor sensation was strong and felt to be unacceptable at the 100
ppm (380 mg/m3) exposure level. Exposures to 10 and 40 ppm (38 and 150 mg/m3) toluene
were without subjective irritation effects. During the 100 ppm (380 mg/m3) exposure, statistically
significant (p < 0.05), but mild irritation was experienced in the eyes and/or nose in 10 of the
subjects; the other six subjects did not report any irritation during exposure to 100 ppm (380
mg/m3) toluene. The irritation was felt just after the exposure began and was constant
throughout the duration of exposure. No irritation occurred in the throat or the lower airways.
About half of the subjects experienced a statistically significant increase in the occurrence of
headaches, dizziness and feeling of intoxication at 100 ppm (380 mg/m3) that was slight to
moderate in intensity. None of the subjects reported nausea or cough.
The subjects in the Andersen et al. study also reported that it became more difficult to
participate in the battery of psychometric tests and that their reaction time felt impaired at 100
ppm (380 mg/m3). In the eight psychometric performance tests covering visual perception,
motor performance, the coordination between visual perception and motor performance,
vigilance, and intellectual capacity, no significant objective changes compared to control
exposures were observed, although there was a borderline significant correlation (0.05 < p <
0.10) for the results of three of the tests – the screw-plate test, Landolt’s ring test, and the
number of errors in multiplication test. Toluene exposure did not cause changes in the nasal
mucus flow, as measured by tagged particle movement from the oropharynx over time, or lung
function tests as measured by forced expiratory vital capacity (FVC), forced expiratory volume in
one second after maximal expiration (FEV1) and in forced expiratory flow during exhalation of
the middle part of FVC (FEF25-75). In addition, nasal flow resistance, as measured by an
oronasal mask with a pneumotachometer, was not affected by toluene exposure. For this study,
the authors concluded that pure toluene up to 100 ppm (380 mg/m3) is only slightly irritating to
the mucous membranes, reduces perceived air quality, is moderately odorous, and causes
some minor, nonsignificant changes in performance.
Dick et al. (1984) examined the comparative psychomotor effects of methyl ethyl ketone and
toluene individually and in combination. Female and male subjects (age = 18-38 years, n
unstated) from college campuses were tested for their ability to pass an “extensive” physical
exam that included neurological screening. Subjects were exposed in a chamber to air, which
included a 2-minute 25 ppm (94 mg/m3) “charge” of a toluene “placebo” exposure (n = 12), or
100 ppm (380 mg/m3) toluene (n = 30 or 32) nominally, for 4 hours. The subjects were tested on
their performance in three tasks before, during, and/or after (up to 1.2 hours post-exposure) the
exposures. There was no true control group breathing plain air in this study. Tasks included
Choice Reaction Time, visual-vigilance, and pattern recognition tests of alertness and
psychomotor function. Subjects also participated in body burden measures of toluene via breath
samples at the following timepoints: 1) the end of the pre-exposure period; 2) 1-2 hours into the
exposure period; 3) at the end of the exposure period; and 4) 90 minutes post exposure. While
the authors stated that pre-exposure tests provided baseline data for comparison with other
periods, it was not apparent that this was done for toluene. Unstated univariate tests and
multivariate analysis of covariance (MANCOVA) were performed.
Results indicated that toluene concentration averaged 100.1 ± 2.5 ppm, range 96.4 ± 7.9 to
102.1 ± 0.9 ppm (379 ± 9.4 mg/m3, range 363 ± 30 to 384 ± 3.4 mg/m3) over the 4-hour
exposure period. Breath toluene levels averaged 24 ± 5.6 mg/m3 (6.5 ± 1.5 ppm) over the 4-
hour exposure period, and were 18 ± 4.9, 26 ± 8.3, 29 ± 4.9, and 29 ± 3.4 mg/m3 at timepoints
1-4, respectively. The authors stated that under constant exposure conditions, toluene breath
concentrations reach steady state within two hours and remain relatively fixed at 10-20% of the
exposure chamber concentration. Psychomotor tests showed that although Visual-vigilance and
pattern recognition test scores trended downward in the 100 ppm (380 mg/m3) group versus the
placebo, with the percentages of correct hits differing most between groups during the first two
hours of exposure, only the Visual-vigilance test yielded a statistically significant difference (p =
0.01 via Manacova analysis). Despite this result, the report concluded it was unlikely toluene
produces biologically significant cognitive decrements at 100 ppm (380 mg/m3). The authors
stated that 100-ppm toluene effects in this study were marginal at most.
Two groups of middle-aged workers (43 men per group) were recruited for an acute toluene
exposure study. Group 1 were printers with occupational exposure to solvents (mainly ethanol,
ethyl acetate, and ethoxyethanol, with small amounts of toluene); Group 2 did not have similar
solvent exposures (Baelum et al., 1985). Forty-one subjects (20 from Group 1 and 21 from
Group 2) were exposed once to 100 ppm (380 mg/m³) of toluene for 6.5 hours while 45 subjects
(23 from Group 1 and 22 from Group 2) were exposed to clean air. No masking agent was used
to disguise the odor of toluene. The subjects completed questionnaires to grade subjective
symptoms before exposure, at onset of exposure, and at the end of exposure. Ten different
performance tests were used to measure psychomotor skills, perceptual skills and vigilance. Six
tests were on different aspects of visuomotor coordination, while the other four were on
perceptual speed and quality, and higher cortical functions. The results were evaluated using an
analysis of variance with p < 0.05 used as the level of significance.
Toluene exposure had an effect on only one of the six visuomotor coordination tests; toluene-
exposed printers (Group 1) were significantly slower in completing the peg board test compared
to printers exposed to clean air. However, no effect was seen between toluene exposed controls
and clean air controls (Group 2). Visual perception in the Landolt’s ring test was significantly
affected in toluene-exposed printers compared to printers exposed to clean air. Again, no effect
was seen between toluene exposed controls and clean air controls. In the color discrimination
test, printers initially performed better overall due to their professional training and education.
However, the control groups had a reduced number of errors in the second test during the day,
while the toluene groups did not improve (p < 0.01). The authors concluded that toluene caused
decreased psychometric performance, mainly in visual perceptual speed and accuracy, with a
tendency toward greater sensitivity in printers.
For the subjective symptoms in the Baelum et al. study, Group 2 workers exposed to toluene
experienced statistically significant eye, nose and throat irritation with exposure to toluene,
which was immediate and remained almost constant throughout the exposure. In Group 1
workers exposed to toluene, only irritation of the nose was statistically significantly increased
with toluene exposure. The subjective data were presented only in graphical form as percent
responding; no absolute numbers of subjects responding or values of percent of subjects
responding were provided. Among other subjective complaints, poor air quality and a strong
odor were experienced immediately among the toluene-exposed workers, but declined towards
the end of exposure. Fatigue ratings were increased significantly at the end of exposure in both
toluene-exposed groups. Feeling of intoxication increased significantly in both exposed groups
throughout exposure.
Olson et al. (1985) exposed 16 healthy men to either 0 or 300 mg/m3 (80 ppm) toluene in a
chamber for four hours to test for nervous function deficits and subjective effects. The
performance tests were administered when subjects first entered the chamber, after exposure
for 2 hours, and after 4 hours of exposure. Analysis of variance was used to determine
statistical significance (p < 0.05) of the performance tests. Isoamyl acetate was administered in
the chamber during the control exposures to disguise the absence of solvent. However, 12 of 16
subjects were able to identify the control condition. The performance on the tests, including
simple reaction time, memory reproduction and choice reaction time, was unaffected by toluene
exposure. Subjective ratings for sensory irritation, headache, nausea, tiredness, feeling of
stress and ability to concentrate were also unaffected by toluene exposure.
A battery of neurobehavioral and performance tests were conducted among 42 young men and
women (21 men and 21 women) exposed to 0, 75, and 150 ppm (0, 280, and 560 mg/m³)
toluene for 7 hours in a chamber (Echeverria et al., 1989). Menthol was used as a masking
agent to disguise the odor of toluene. Fourteen subjects were randomly assigned to each of the
three groups and were exposed to one of the above 3 concentrations on each day of the 3 days
of testing. A 3 × 3 Latin square study design was employed, so each group of 14 subjects was
exposed to a different order of toluene concentrations. A 5-10% decrement in performance was
considered significant if it was consistent with a linear trend at p < 0.05. Tests were conducted
daily before, during and after exposure where each subject served as their own control.
Scheffe’s 95% confidence intervals were used to identify significant differences in scores
between the control and exposed groups.
With these criteria, Echeverria et al. found statistically significant linear trends for decrements in
verbal short term memory (digit span test, 6.0% decrement), visual pattern memory (number
correct, 5.0% decrement), visual perception (pattern recognition latency, 12.1% decrement),
psychomotor skills (critical tracking test, 3.0% decrement), and manual dexterity (one hole test,
6.5% decrement), with a statistically significant score difference between 0 and 150 ppm (570
mg/m3) exposures. A significant difference was also noted between the 0 and 75 ppm exposure
groups for the pattern recognition latency test. No gender-related differences in the
neurobehavioral and performance tests were observed.
Echeverria et al. also noted reports of subjective symptoms of headache (19, 26, and 33% at 0,
75, and 150 ppm, respectively), eye irritation (17, 21, and 48% at 0, 75, and 150 ppm,
respectively), and number observed sleeping during exposure (7, 14, and 22% at 0, 75, and 150
ppm, respectively) that increased with increasing dose. The dose-dependent increase (p <
0.001) in the number of observations of subjects sleeping during the exposure was concluded
by the authors to be the most convincing evidence of toluene affecting the central nervous
system. Although statistical analysis between dose levels was not provided for subjective
symptoms by the authors, the data suggest a statistically significant increase in headache and
eye irritation at 150 ppm (570 mg/m3).
Half the subjects correctly guessed their order of exposure despite the use of menthol as a
masking agent. However, a comparison of performance between the successfully blinded
subjects and the non-blinded subjects showed no significant differences. The authors concluded
that toluene had significant but small acute behavioral effects mostly in the range of 2 to 7%
performance decrements at 150 ppm (570 mg/m3).
Baelum et al. (1990) conducted another chamber study in healthy adults (32 males and 39
females) in which exposures were to a constant concentration of 93 ppm (350 mg/m3) toluene,
or a fluctuating concentration of toluene between 300 and 50 ppm (TWA of 102 ppm, 380
mg/m3) for 7 hours. Specifically, fluctuating exposures consisted of 14 episodes of 30 min with
an increasing concentration reaching a peak of 300 ppm (1100 mg/m3) after 5 min, then
decreasing to 50 ppm (190 mg/m3) over 10 min where the concentration remained for another
15 min. The subjects were divided into three groups of 23-24 in which each group had a single
exposure to either control, constant toluene concentration, or the varying toluene concentration.
Three periods of exercise on an ergometer cycle for 15 min were conducted during each
exposure, with workloads of 40 and 60% of maximal aerobic capacity. Subjects exposed to the
varying concentration of toluene were exercised during peak concentrations. Subjective ratings
and four performance tests (peg board, color test, vigilance clock test, five-choice serial reaction
test) were conducted twice during exposure. Standard analysis of variance was used to test
each variable using p < 0.05 as the measure for statistical significance.
For subjective findings, bad air quality, odor level, irritation of nose and lower airways, feeling of
intoxication and dizziness were greater in the toluene exposed groups compared to the control
group (Baelum et al., 1990). There was no difference between the two exposed groups. No
differences between any groups were observed for the performance tests, although a tendency
(0.05 < p <0.10) towards lower score and more errors in the vigilance test was found in the
toluene exposed groups. The only apparent gender difference observed was that females
worked faster on the peg board test and performed better on the color test compared to males.
The authors concluded that the subjective sensory irritation and light neurotoxic symptoms
occurred with toluene exposure in accordance with their previous study, while only a weak
indication in performance tests was observed, unlike their previous study.
Echeverria et al. (1991) compared the abilities of toluene and ethanol to produce non-specific
depressive CNS effects in human volunteers. Only experiments involving toluene are discussed
herein. Paid subjects (n = 14/group; male and female; age 18-35 years, ≥ 1 year college
education) were exposed to toluene (0, 282, and 564 mg/m3, or 0, 75, and 150 ppm) and
menthol (an odor masking agent; 0.29 mg/m3 or 0.078 ppm) for seven hours/day over three
days. The subjects were evaluated on their performance in several behavioral tests that
measured short-term pattern memory (Sternberg, digit span, and Benton tests); perception
(pattern recognition test); psychomotor skill (simple reaction time, continuous performance,
symbol-digit, eye-hand coordination, finger tapping, and critical tracking tests); manual dexterity
(one hold test); mood [profile on mood scales (POMS)]; fatigue (fatigue checklist); and verbal
ability. Voluntary subject observations regarding sleep and exposure-related ailments were also
noted. Each day, pre- (baseline control) and post-exposure test performance were measured at
08:00 and 16:00, respectively. Toluene exposure concentrations varied for each group on a
daily basis such that all subjects experienced every exposure condition by the end of the study
(one exposure concentration/day).
According to the study authors, complaints of eye irritation and headaches increased in a dose-
dependent manner. However, the authors did not provide a quantitative estimate of these
effects. At the highest exposure concentration, notable effects included performance
decrements in digit span (-6%), pattern recognition (-12%), pattern memory (-5%), one hole (-
5%), and critical tracking (-3%) tests with ANOVA analysis. By grouping males and females
together for the study, the authors imply that there were no gender differences in responses to
the neurobehavioral tests. However, the authors did not explicitly state this.
Rahill et al. (1996) conducted an assessment of physical and neuropsychological performance

during low-level exposure to toluene. Healthy, non-smoking male (n = 2) and female (n = 4)
Caucasian volunteers between the ages of 27 and 38 years were exposed to conditioned room
air or 380 mg/m3 (100 ppm) of toluene for 6 hours in an exposure chamber in a double-blind,
randomized manner. Each subject served as his/her own control, as the experimental design
involved a two-period crossover with at least 14 days separating the two successive exposure
periods.
Subjects exercised for 30 minutes on a bicycle ergometer during the exposure period to
approximately quadruple their resting ventilation. Respiratory function was measured before and
immediately after exposure, and levels of toluene in blood and exhaled breath were measured
before, twice during, immediately after, and 1 hour and 2 hours after exposure, respectively.
Two computerized neuropsychological tests were performed in three repetitions: a brief
standard battery, the Automated Neuropsychological Assessment Metric (ANAM), which lasted
20 minutes and required the subject to respond to 7 screen tests presented individually, and an
hour-long multitask performance test using the SYNWORK system that required the subject to
respond to 4 simultaneous work windows on the computer screen.
Results showed an increase in absorption of toluene during exercise, with mean blood and
exhaled air samples averaging 1.5 µg toluene/g blood and 28 ppm (104 mg/m3) of toluene,
respectively. No significant changes were observed in lung function following toluene exposure
relative to baseline or exposure to conditioned air. Exposure to toluene was found to
significantly alter both performance and response time on neuropsychological tests: overall
performance (Composite score) in the prolonged multitasking test declined during toluene
exposure relative to the room air condition (F = 29.2, p = 0.005), with a 10% reduction during
the 6th hour of toluene exposure, and the mean response time indicated by the ANAM battery
was significantly longer for toluene exposure than for conditioned air. Further, the Composite
score improved with exercise under the room air condition but remained flat throughout the
exposure period to toluene. No apparent gender-related differences were observed, although
the number of subjects were too small for statistical verification.
Orbaek et al. (1998) examined subjective reactions to toluene and n-butyl acetate among
subjects with chemical sensitivity. Two exposure groups were used. The first group employed
subjects with symptoms and neuropsychological test results compatible with toxic
encephalopathy (TE) type 2A (TE-2A) and 2B (TE-2B) following long-term occupational
exposure to solvents. The second group (controls) consisted of previously unexposed
referents. All subjects were men between the ages of 28 and 66, and each group consisted of
12 subjects. Both groups were exposed over a period of 70 minutes in exposure chambers to 11
to 180 mg/m3 (3 to 48 ppm) toluene, which is below previously reported thresholds for acute
neurotoxic effects and trigeminal irritation (750-1125 mg/m3, 200-300 ppm).
The test results showed that the TE groups were more sensitive to toluene exposure than the
referents. Ratings of smell intensity, which was measured on a 7-step category scale from none
to extremely strong and represented by regression coefficients (mean ± SD), were higher for the
TE-2B group (1.05 ± 0.12) than for the TE-2A (0.73 ± 0.32) and referent (0.79 ± 0.15) groups
during toluene exposure (p < 0.001). Mean regression coefficients for mucous membrane
irritation from toluene exposure, which was rated on a visual analogue scale from 0 to 100, were
higher for the TE-2A (7.8 ± 5.0, p = 0.009) and TE-2B (7.8 ± 5.5, p = 0.008) groups than for the
referent group (2.1 ± 2.4). Both TE groups had higher ratings for fatigue from toluene exposure
than the referent group: mean regression coefficients were 1.0 ± 1.5 for the referent group and
7.8 ± 6.1 and 6.8 ± 5.7 for the TE-2A and TE-2B groups, respectively (p <0.001).
Little et al. (1999) studied the effects of toluene on human subjects clinically sensitive to
toluene. Twenty toluene-sensitive patients (n = 11 females, 9 males; average age = 39.5 years)
were exposed in a specially designed testing booth to 15 ppm (56 mg/m3) toluene for 20
minutes along with 16 reportedly healthy controls who had no history of adverse chemical
exposure (n = 8/sex; average age = 32 years). The subjects were assessed through
neurobehavioral and/or blood chemistry tests before and after the toluene exposure. The battery
of neurobehavioral tests evaluated visual acuity (Focal Length), immediate and delayed verbal
memory recall (Babcock stories; prose memory), information processing time (Simple Reaction
Time; SRT), concentration and attention (Letter Cancellation and STROOP Color Word), and
psychomotor coordination (Symbol Digit ). Blood serum was used to determine whether toluene
exposures resulted in B- or T-cell-mediated immune responses. To this end, levels of B-cell-
derived immunoglobulins (IgGs) and T-cell-derived antigen-specific binding molecules (TABMs)
were measured via an enzyme-linked immunosorbent assay (ELISA).
Results indicated that the toluene-sensitive patients had moderate to severe clinical reactions
with typical symptoms including fatigue, poor concentration, headache, and muscle pains upon
exposure to toluene. Neuropsychological testing revealed worsening performance on post-
versus pre-exposure Babcock (p ≤ 0.009), Symbol Digit (p = 0.0099), and Letter Cancellation (p
= 0.0380) tests. Patients exhibited significantly (p = 0.002) greater numbers of TABMs to the BA
HSA antigen compared to controls. The study results suggested that acute, low-concentration
(15 ppm; 56 mg/m3) toluene exposure produced deficits in memory, coordination, and mental
focus with primarily T- versus B-cell mediators in sensitive individuals. In this study, 15 ppm is a
LOAEL. The occurrence of any potential gender-specific differences in neurobehavioral test
results was not addressed by the authors.
Osterberg et al. (2000) evaluated psychological test performance of chemically sensitive human
subjects exposed to toluene or n-butyl acetate. This publication supplements the previous one
of Orbaek et al. (1998) described above, providing additional data from neuropsychological test
results collected during the exposure chamber sessions described by Orbaek et al. (1998) for
the same study population. Overall, results from the three psychological performance tests
administered (the digit symbol test, the Automated Psychological Test System [APT] two-way
reaction-time test, and the APT inhibition test) showed that the TE subjects were not more
affected than healthy reference subjects by toluene exposures at the test concentrations of 11
to 180 mg/m3 (3 to 48 ppm).
Osterberg et al. (2003) studied subjective responses and psychological test performance of
chemical-sensitive human subjects upon exposure to toluene or n-butyl acetate. Ten women
with symptoms compatible with multiple chemical sensitivity (the test group) and 20 healthy
women (the control group) were exposed to five concentrations of toluene, from 11 to 180
mg/m3 (3 to 48 ppm), over a period of 70 minutes in exposure chambers. The results showed
that there were steeper increases of ratings for mucous membrane irritation and fatigue in the
test group than the control group, while the ratings of smell intensity and smell annoyance were
similar in the two groups. For the three psychological performance tests (the digit symbol test,
the Automated Psychological Test System [APT] two-way reaction-time test, and the APT
inhibition test), while reductions in test performance were observed in both groups, the decline
observed in the test group was more prominent (p < 0.05).
To study the influence of the inhalation exposure pattern on the toxic effect of toluene, Lammers
et al. (2005) designed a human volunteer study to compare the neurobehavioral effects of
exposure to regularly occurring peak concentrations and constant exposure at the same
average level. Eleven healthy men (age 20-49 yrs) were exposed for 4 hr on two different days
separated by a seven-day wash-out period. One exposure was to a constant concentration of
40 ppm (150 mg/m3) toluene; the other exposure was to a time-weighted average dose close to
40 ppm, but included three 30-min exposures to a peak concentration of 110 ppm (410 mg/m3).
The results showed no clear changes in neurobehavioral function, including tests of motor
performance, attention, perceptual coding, and memory. No clear changes in mood and effect
were observed. The authors concluded that these conditions did not induce significant acute
changes in central nervous system function as shown at much higher concentrations in animals.
Visual effects
The Baelum et al. (1985) study described above, where middle-aged workers (43 per group)
were recruited for an acute toluene exposure study (Group 1 had previous occupational
exposure to solvents and Group 2 did not), also included an evaluation of color vision effects
resulting from toluene exposure. The study results showed statistically significant decrements in
both groups for color discrimination and accuracy in visual perception with the Landolt’s ring
test. A statistically significant decrease in visuomotor function was observed with the peg board
test, but the effect was restricted to toluene-exposed workers. In addition, a trend (0.05 < p <
0.10) toward decreased vigilance with the lamp test was observed in both exposed groups. The
authors concluded that acute exposure to 100 ppm (380 mg/m3) toluene adversely affects
persons irrespective of former occupational solvent exposure.
The effects of acute toluene exposure on color vision were studied in a group of eight
rotogravure printing workers that had been employed and occupationally exposed to toluene for
an average of 9.8 years (Muttray et al., 1999). In rotogravure printing, toluene is generally the
only solvent used. The color vision acuity of the workers before and after an acute toluene
exposure (28 – 41 minutes in duration, 300-400 ppm, 1100 – 1500 mg/m3 of toluene) was
evaluated using the Farnsworth panel D-15 test, the Lanthony desaturated panel D-15 test, and
the Standard Pseudoisochromatic Plates test part 2. A control group of 8 unexposed workers
(from a metal-working factory) was also tested. One exposed worker left the room due to
headache. Acute exposure to toluene did not impair color vision or cause other narcotic
symptoms. Print worker performance prior to acute toluene exposure was similar to controls on
the Farnsworth panel D-15 and Standard Pseudoisochromatic Plates part 2 tests. However,
print worker performance on the Lanthony desaturated panel D-15 test prior to exposure was
slightly worse (suggesting chronic effects) than that of controls, with median scores of 1.18 and
1.05 for exposed and controls (higher number indicates degraded performance), respectively.
The difference was of borderline statistical significance (p = 0.06). The authors noted that the
small number of subjects (n = 8) limited the statistical power of the study.
The Baelum et al. (1990) study described above also included a color test to evaluate the effect
of 7-hour, TWA 100 ppm (380 mg/m3) toluene exposure on visual performance for 32 males and
39 females from a random sample of the general population. In the color test the subjects
compared a list of 20 color samples with a reference table (20 color specimens test). The
number of correct answers together with the cumulated deviation according to color and
intensity were used as score. The result showed no effect of toluene exposure in color test
results, indicating only minimal if any effect on visual performance. However, as noted above,
females performed better overall on the color test compared to males.
Muttray et al. (1995) examined the subacute effect of toluene on color vision in 59 rotogravure
workers (mean age 36 yrs, mean rotogravure working duration = 10 yrs) without a control group.
The work-shift exposure was analyzed by taking venous blood samples immediately before
color vision testing and measuring both toluene and ethanol (a potential confounder)
concentrations. However, the authors did not measure workplace air toluene concentrations.
Color vision tests were performed on Monday before shift following 2 to 3 days of exposure-free
time, and on Friday after shift. Color vision tests used included the Ishihara plates, the Velhagen
plates, the standard Pseudoisochromatic Plates part 2, the Farnsworth panel D-15 test and the
Lanthony desaturated panel D-15 test. The average toluene concentration in blood ranged from
< 0.22 to 7.37 mg/L, and the color vision tests did not show any influence of toluene
concentration on the color confusion indices. The authors concluded that there was no increase
in acquired color vision impairment over the work week with toluene exposure partially
exceeding 1.7 mg/L (threshold for biomonitoring in Germany).
Both human and animal studies revealed that acute toluene exposure can disturb different
neurotransmitter systems. To investigate whether the visual attention processes is a target of
toluene in humans, Kobald et al. (2015) applied a visual change detection task to 17 young
healthy human volunteers (mean age of 24.12 yr), using electroencephalography (EEG) to
measure neurobehavioral and neurophysiological effects of a single peak exposure of 200 ppm
(750 mg/m3) toluene for 40 min at light physical activity to mimic a real-life working situation,
with 16 volunteers (mean age of 25.25 yr) as control group. The behavioral results showed that
toluene impairs the rate of correct responses especially in task conditions in which an irrelevant
distractor is given, while the response times did not differ between the experiment and control
groups. The neurophysiological results implied a less efficient visual processing of relevant
stimuli and an increased distractibility by irrelevant stimuli.
Pulmonary effects
Stewart et al. (1975) exposed groups of male subjects in a chamber to a different concentration
of toluene (0, 20, 50 or 100 ppm, 0, 80, 190 or 380 mg/m3) each week over four-week period.
Three different groups of 2-4 subjects each, consisting of 1-, 3-, and 7.5-hour exposure groups,
were exposed each day for up to 5 days per week. During the fifth week, three groups of male
subjects (2-4 subjects per group), divided into 1-, 3-, and 7.5-hour exposure groups, were
exposed to a fluctuating concentration of toluene between 50 and 150 ppm (190 and 570
mg/m3) each day for 5 days. In addition, three groups of female subjects (2 or 4 subjects per
group) were divided into
1-, 3- and 7.5-hour exposure groups and exposed to 100 ppm (380 mg/m3) toluene each day for
5 days. Pulmonary function tests (forced expiratory vital capacity [FVC], forced expiratory
volume in one second after maximal expiration [FEV1], peak expiratory flow rate [PEFR] and
flow rate at 50% of FVC [MMEF]) on two subjects per group in the Stewart et al. (1975) study
did not reveal any changes due to the toluene exposures. Likewise, regulation of ventilation,
heart rate and alveolar gas exchange in the subjects examined (n = 4) were unaffected by
toluene exposure.
Renal effects
Nielson et al. (1985) investigated the renal effects from acute exposure to toluene in the same
groups of workers exposed to toluene by Baelum et al. (1985). The exposure protocol of 0 and
100 ppm (0 and 380 mg/m3) toluene for 6.5 hours is described in detail above in Baelum et al.
(1985). Changes in the excretion rate of albumin and ß2-microglobulin were used as indicators
of glomerular and renal tubular damage, respectively. No significant changes in renal excretion
rates of either protein were found following acute exposure to 100 ppm (380 mg/m3) toluene
suggesting that acute exposure to toluene has no nephrotoxic effect by these measures.
Other Studies
There are a number of older human inhalation studies of varying quality investigating the acute
toxicity of toluene. The following human studies support the association between toluene
exposure and the known acute effects. However, the value of these studies is limited by issues
such as poorly described or unconventional health endpoints, inadequate descriptions of the
methodology, and questionable toluene exposure concentrations used.
Male volunteers were exposed to toluene via mouthpiece in which the concentration increased
in a stepwise fashion from zero to 900 mg/m3 (240 ppm) over 40 minutes (Horvath et al., 1981).
The concentration was then held at 240 ppm for 30 min for a total exposure time of 70 min. The
air was perfumed presumably to hide the odor of toluene. A total of 23 subjects were exposed,
approximately half of which received a capsule containing diazepam, and the other half a
placebo. These groups were compared to groups given only the placebo or diazepam. Testing
for alertness consisted of spatial discrimination of acoustical clicks and a continuous visual
feedback task. No decrement in vigilance performance was observed during exposure in the
toluene + placebo group, although the toluene + diazepam group showed a worsening of
performance (p < 0.05). However, both toluene exposure groups showed a significant decrease
in performance when measured 70-140 min following cessation of toluene exposure.
In order to elucidate the mechanism of toluene reproductive toxicity, Luderer et al. (1999)
studied the reproductive endocrine effects of toluene acute exposures in healthy human
subjects 10 males and 20 females aged 19-45 yrs. Women were divided into two groups those
in follicular phase and those in the luteal phase of the menstrual cycle. A 3-hour exposure to 50
ppm (190 mg/m3) toluene through a mouthpiece did not result in alterations of their plasma
luteinizing hormone (LH) or follicle stimulating hormone (FSH) secretion profile. However, subtle
effects on LH secretion were identified, a greater decline in LH pulse frequency for women in
the luteal phase (p = 0.06) and a greater LH decline in men (p < 0.05) than their respective
control groups. There was no effect on blood testosterone levels in men. The authors concluded
that the clinical relevance of the subtle effects on LH secretion was unclear.
The controlled human acute exposure studies with known exposure concentrations are
summarized in Table 1.
Table 1. Summary of the principal studies for the acute toxicity of toluene in human adults.
Exposure duration NOAEL LOAEL
and Concentration ppm ppm
Study (ppm) Subjects / Effects (endpoints) (mg/m3) (mg/m3)
Von 8 hr at 50, 100, 3 healthy human beings 100 200

Oettingen 200, 300, 400, 600, Minor symptoms of fatigue, headache and (380) (750)
et al. and 800 ppm drowsiness at 50 and 100 ppm.
(1942)
Impairment of coordination and reaction time
beginning at 200 ppm.
Longley ~ 30 min at 29 (accident 1), 7 (accident 2) * 10,000
(1967) estimated 10,000- Euphoria, drunkenness, dizziness, nausea, (38,000)
30,000 ppm loss of consciousness, confusion,
incoordination, drowsiness
Gamberal 20 min at 100, 300, 12 healthy males. 100 300
& 500, and 700 ppm Impaired simple reaction time beginning at (380) (1,100)
Hulttengren with 5 min break 300 ppm.
(1972) between 300 and
500 ppm Impaired perceptual speed at 700 ppm.
Stewart et 1-hr, 3-hr, and 7.5- 2-4 males, and 2-4 females per group 50 100
al. (1975) hr per day At 100 ppm: Eye, nose and throat irritation in (190) (380)
exposures, up to 5 3-hr group only. Inflamed nostrils in 7.5-hr
d/wk. group on day 5 of exposure.
Males: 0, 20, 50, Change in visual evoked response in one
100 ppm, and subject.
variable 50-150
ppm groups Increased errors in alertness in 7.5-hr females
Females: 0 and 100

ppm groups.
Horvath et 3 30-min sessions 11 males age 20-21. * 240
al. (1981) at 240 ppm Impaired vigilance during third session (900)
Andersen 6 hr at 0, 10, 40, 16 young healthy males 40 100

et al. and 100 ppm At 100 ppm: increased eye and/or nose (150) (380)
(1983) irritation (10 of 16); headache, dizziness, and
feeling of intoxication.
Significant correlation for increased odor and
bad air with increasing toluene concentration.
Borderline correlation (0.05 < p < 0.1) for
decrement in 3 of 8 psychometric
performance tests.
No effect on nasal flow resistance, nasal
mucus flow and lung function.
Abbreviation: * not observed.
Table 1. Summary of the principal studies for the acute toxicity of toluene in human
adults (continued).
Dick et al. 2-min (placebo) 12 (placebo) or 30-32 males and females * 2-min
(1984) exposure at 25 ppm age 18-38 yrs (placebo)
or 100 ppm for 4 exposure
hours at 25 ppm
or 100
ppm for 4
hours
Olson et al. 4 hr at 80 ppm 16 males age 23-38yr 80 *
(1985) No effect on subjective symptoms; no (300)
impairment in 3 psychomotor tests
Baelum et 6.5 hr at 0 ppm (n = G1 = 43 workers with previous solvent * 100
al. (1985) 23 G1 workers and exposure.G2 = 43 workers with no previous (380)
22 G2 workers) or solvent exposure.
100 ppm (n = 20 At 100 ppm:
G1 workers and 21
G2 workers). Subjective results: Increased eye (G2 only),
nose, and throat (G2 only) irritation,
increased strong odor, and low air quality,
increased feeling of fatigue and intoxication.
Objective findings: decreased color
discrimination, visual perception (Landolt’s
ring test), and visuomotor function (peg
board test, G1 only).
Nielsen et Same as Baelum et Same as above. 100 *
al. (1985) al. (1985) At 100 ppm, no significant changes in (380)
excretion rate of albumin and ß2-
microglobulin, no nephrotoxic effect.
Echeverria 7 hr at 0, 75, and 42 young men & women 75 150
et al. 150 ppm Neurobehavioral results: At 75 ppm – (280) (570)
(1989, decreased visual perception.
1991)
At 150 ppm – Decreased verbal short term
memory, visual pattern memory, visual
perception, and manual dexterity - all
showing a linear trend.
Objective findings: Dose-response increase
in headache, eye irritation and sleeping
episodes with increasing concentration.
Table 1. Summary of the principal studies for the acute toxicity of toluene in human adults
(continued).
Baelum et 7 hr at 0, 93, and 32 males 39 females age 31-50. * 100

al. (1990) TWA 103 ppm (50 (380)
ppm with 300 ppm For both toluene exposure groups: Increased
peaks) subjective response to odor, bad air, irritation
of nose and lower airway, feeling of
intoxication and dizziness. Decreased
performance in one (vigilance test) of four
psychomotor tests (0.05 < p < 0.10).
Little et al. 20 min at 15 ppm 20 toluene-sensitive patients (9 male, 11 * 15 (56)

(1999) female).
Moderate to severe clinical reactions with

symptoms including fatigue, poor
concentration, headache, and muscle pains;
worsened neuropsychological test
performances after 15 ppm toluene exposure.
Luderer et 3 hr at 50 ppm 10 male 20 female age 19-45. 50 *

al. (1999) (190)
No change in plasma LH or FSH secretion
profile; greater decline in LH pulse frequency
for women in the luteal phase, and a greater
LH decline in men.
Muttray 28-41 min at 200- 8 toluene-exposed workers, 8 controls. 400 *

(1999) 400 ppm (1500)
No effect on color vision at test exposure in
either group.
Borderline impairment (p = 0.06) of color

vision in toluene-exposed workers prior to test
exposure.
Lammers 4 hr at 40 ppm. 11 healthy adult males age 20-50. 40 *

et al. (150)
(2005) (TWA ~40ppm) No changes in neurobehavioral tests of motor
plus 3 30-min performance, attention, perceptual coding,
peaks of 110 ppm. and memory, and no changes in mood.
Acute Toxicity to Infants and Children

Embryopathy due to toluene exposure was first suggested in 1979 in an infant with phenotypic
features similar to the fetal alcohol syndrome (FAS), born to a woman who abused toluene-
based solvents during pregnancy (Toutant and Lippmann, 1979). Since that time, similar cases
resulting from toluene-based solvent abuse have been reported in the literature.
Hoyme et al. (1993) reported 12 children whose mothers abused toluene-based spray paint by
inhalation during pregnancy. Fifty-eight percent had intrauterine growth retardation, and 3 of 4
followed beyond the neonatal period showed postnatal growth deficiency. Seventy-five percent
had craniofacial features consistent with FAS. Three of the 12 had hydronephrosis. Analysis of
the pattern and nature of associated malformations suggests a common mechanism of
teratogenesis for toluene and alcohol, namely, a deficiency in craniofacial neuroepithelium and
mesodermal components due to increased embryonic cell death.
The brain continues to develop in children through adolescence. As a result, children are likely
to be more susceptible to the neurotoxic effects of toluene due to less cell differentiation and
increased cell division compared to adults (OEHHA, 2008). Undifferentiated cells tend to be
more prone to injury than differentiated cells. As will be summarized below, animal studies have
shown that the young are more susceptible to the neurotoxic effects of toluene compared to
adults.
As indicated in Section 4, infants under one year of age may not metabolize toluene to benzyl
alcohol as quickly as adults. The parent compound, toluene, is thought to be the main form that
results in toxic effects. Therefore, slower metabolism of toluene may result in longer, more
potent deleterious effects in exposed infants.
Acute Toxicity to Experimental Animals
Neurobehavioral Effects
Dose- and age-dependent decreases in behavioral performance and depression of the central
nervous system were observed in mice and rats exposed by inhalation to toluene at
concentrations ranging from 2600 to 12,000 ppm (9800 to 45,000 mg/m³) for up to 3 hours
(Bruckner and Peterson, 1981). Younger animals were more susceptible to toluene toxicity and
mice were more sensitive than rats of the same age (statistical analysis not performed by
authors). Four-week-old mice were depressed more rapidly than were 8- and 12-week-old mice
exposed to toluene concentrations of 2600, 5200, and 12,000 ppm (9800, 19,600 and 45,000
mg/m3). Four-week-old rats were also slightly more sensitive than were older animals. Although
mice and rats were narcotized similarly after 2 to 3 hr of toluene inhalation, mice appeared to
succumb more rapidly than did rats of the same age.
To assess how behavioral effects differ in adolescents compared to adults, Batis et al. (2010)
exposed 72 adolescent (postnatal day [PN] 28) and 72 adult (PN 90) male rats to toluene for
two 15-min durations separated by a 120-min interval (30 min/day) over 12 days at
concentrations of 0, 8000 or 16,000 ppm (0, 30,000 or 60,000 mg/m3). Locomotor activity was
measured during toluene exposures and 30 min after the final daily exposures. Compared to
adults, adolescents displayed greater locomotor activity on the first day and generally greater
increases in activity over subsequent days during toluene exposure. Adults showed greater
locomotor activity than adolescents in the “recovery” period following exposure on the first and
subsequent days. The results are consistent with dose-dependent shifts in sensitivity and
sensitization or tolerance to repeated toluene in the adolescent animals compared to the adult
animals.
Batis et al. (2010) noted that toluene and many of the other commonly abused volatile organic
solvents resulted in concentration-dependent effects on activity in adult rodents characterized
as progressing from initial and low-dose motor excitation, then to sedation, motor impairment
and anesthesia as concentration and duration of exposure increase. In rodents, the authors
pointed out that these results are in general agreement with previous studies using adult rats
(Hinman, 1987; Yavich et al., 1994) and adult mice (Bowen and Balster, 1998; Wood and
Colotla, 1990) in which the acute locomotor effects of inhaled toluene were shown to be
biphasic, with excitation at concentrations below 4000 ppm and sedation with motor impairment
at concentrations above 6000 ppm. Similar biphasic action on locomotor activity was observed
with gaseous anaesthetics and barbiturates (Evans and Balster, 1991). Many animal studies
have described critical periods for cognitive development in the young that would increase their
sensitivity to toluene exposure relative to adults (Kalsbeek et al., 1989; Frohna et al., 1995;
Joyce, 1996; Lipska and Weinberger, 2002; Schwabe et al., 2004). These studies identified the
anatomical areas that send axonal projections and neurotransmitters to connect with those in
other areas of the brain to perform specific cognitive functions. It is reasonable to extrapolate
these results to human children because rats are generally considered good models of both
human brain dysfunction and normal learning processes (Loupe et al., 2002; Smidt et al., 2003;
Vitalis et al., 2005), and rat brain development parallels human brain development in all but
timeline and complexity of the human cortex (Nieoullon and Coquerel, 2003; Juraska and
Markham, 2004; Vidair, 2004). Investigations of the developmental effects of toluene in animal
models are summarized in Section 7, Developmental and Reproductive Toxicity, below.
Other Neurological Effects

The 1-hour LC50 for toluene in the rat was estimated at 26,700 ppm (100,000 mg/m³) (Pryor et
al, 1984). The 6-hour LC50s in rats and mice were 4618 ppm (17,320 mg/m³) and 6949 ppm
(26,060 mg/m³), respectively (Bonnet et al., 1982). An 8-hour LC50 was estimated to be 5300
ppm (19,900 mg/m³) in the mouse. By inhalation, toluene has been reported to be more acutely
toxic in animals than the similar compound benzene (Svirbely et al., 1943).
As evaluated using multisensory Conditioned Avoidance Response (CAR) Task training that
involves the use of behavioral audiometry and electrophysiologic audiometry, hearing loss was
observed in groups of rats after exposure to various exposure scenarios: 1000 ppm (3760
mg/m³) toluene, 14 hours per day for 2 weeks; 1500 ppm (5700 mg/m3) for 14 hours per day for
three days; 2000 ppm (7500 mg/m3) for 8 hours per day for three days; and intermittent
exposure to 3000 ppm (11,000 mg/m3) for 30 minutes every hour, 8 hours per day for 2 weeks
(Pryor et al., 1984). However, groups of rats exposed to single exposures of 4000 ppm (15,100
mg/m3) for 4 hours or 2000 ppm (7500 mg/m3) for 8 hours did not develop ototoxicity.
Kishi et al. (1988) used the shock avoidance response test to study behavioral effects in rats.
Inhalation exposure to 125, 250, or 500 ppm (470, 940 or 1900 mg/m3) toluene for 20 minutes
resulted in decreased conditioned avoidance response that was reversible. However, exposure
to 1000 ppm (3800 mg/m3) toluene for about four hours and 2000 ppm (7500 mg/m3) for two
hours produced a concentration-related increase in incorrect responses and a considerable
decrease in the effective avoidance response rate.
Rogers et al. (1999) examined the effect of neurobehavioral sensitization to toluene in two
groups of 8 rats each, with two other groups of 8 rats each as controls. The first group was
exposed to one acute exposure of 1600 ppm (6000 mg/m3) toluene for 6 hrs on one day (acute
group); the second group was chronically exposed to 80 ppm (300 mg/m3) for 6 hr/day for 4
weeks (repeat group). After 17 days of no exposure, a subsequent very low exposure (10 ppm,
38 mg/m3; termed a triggering dose) was given to see if there were differences in operant
performance between the acute and chronic exposure groups. One of the two control groups
was exposed to 10 ppm (38 mg/m3) toluene as well (trigger group), while the other group was
exposed to clean air. Trigger and sham exposures and operant testing were continued 5
days/week for 17 sessions. The operant response was to press a lever for food the correct
number of times, with the number of lever presses for food changing at specific intervals. The
investigation was duplicated with one replicate of 32 female rats and then another of 32 male
rats. An increased number of incorrect responses, were seen in the acute, repeat, and trigger
groups compared to the control. Both the acute and repeat males and females were adversely
affected by the initial toluene exposure. The females treated with a trigger dose demonstrated
no deficits, but their male counterparts were adversely affected by the trigger exposures.
To evaluate the rewarding effects of toluene inhalation, Funada et al. (2002) put 5 groups of
male ICR mice into an airtight inhalation shuttlebox and tested their toluene inhalation-related
place preference. Conditioning (training) sessions of 20 min each were given twice daily (one
for toluene and one for air) for 5 days, with a minimum of 7 hr between sessions. The 5 groups
of mice were exposed to 0 (control), 350, 700, 2500, or 3200 ppm (1320, 2600, 9400, or 12,100
mg/m3) toluene (measured by GC) in one of the two compartments of the same shuttleboxes,
respectively. Test sessions were one day after the final conditioning session with no toluene
exposure. The time each mouse spent in each compartment during a 20-min session was
measured using a digital video camera. The results showed the exposure to toluene ≥ 700 ppm
(2600 mg/m3) produced a significant place preference (to avoid toluene exposure) in mice. In
this study, 700 ppm (2600 mg/m3) is a LOAEL.
Using well-established pattern-elicited visual evoked potentials (VEPs) and a physiologically

based pharmacokinetic (PBPK) model to estimate the toluene brain concentration during and
after inhalational exposure to toluene, Boyes et al. (2007) demonstrated that toluene impaired
visual function in rats. Adult male Long-Evans rats were exposed by inhalation to 1000 ppm
(3800 mg/m3) toluene for 4 hours, 2000 ppm (7500 mg/m3) for 2 hours, 3000 ppm (11,300
mg/m3) for 1.3 hours, or 4000 ppm (15,100 mg/m3) for 1 hour. Brain neurophysiological function
was measured using VEP recorded from electrodes located over visual cortex of the rats. The
VEP amplitude of the major spectral component was reduced by toluene exposure. The
experiment data showed a logistic fit with a significant correlation between VEP amplitude
reduction and brain toluene concentration. The authors also concluded that the acute neurotoxic
effects of toluene are caused by perturbations of various neurotransmitter systems and ion
channels involved in neurotransmission.
Acute exposure to toluene results in neurotoxicity including alterations in visual function. N-

methyl-D-aspartate (NMDA)-glutamate receptors are widely present in the visual system and
contribute to pattern-elicited VEPs in rodents. To elucidate the mechanisms underlying the
visual neurotoxicity of toluene, Bale et al. (2007) studied whether acute toluene effects on
NMDA-glutamate receptors contribute to toluene-induced alterations in VEPs of rats. Long-
Evans rats were exposed to 2000 ppm (7500 mg/m3) toluene by inhalation, and VEPs were
measured during toluene exposure in the presence or absence of NMDA (agonist) or MK801
(antagonist). The results showed that the amplitude of VEPs, which strongly rely on
glutamatergic neurotransmission, decreased after the exposure to toluene and that this effect
could be reduced by pre-administration of MK801. The authors claimed that the data support
the hypothesis that especially early visual processing is partly inhibited by toluene.
Bowen et al. (2010) compared four mouse strains (three inbred strains, Balb/cByj, C57BL/6J
and DBA/2J, and one outbred strain, Swiss Webster) of five groups in their sensitivity to
changes in locomotor activity following acute exposure [30 min, 0, 100, 2000, 8000, and 10,000
ppm (0, 380, 7500, 30,000 and 38,000 mg/m3)], and then each group with repeated [8000 ppm
(30,000 mg/m3), 30 min/day for 14 consecutive days] toluene exposure. With acute exposure,
qualitatively similar acute behavioral effects were observed in all four strains of mice, in that a
biphasic effect on spontaneous locomotor activity was produced in the mice. At 2000 ppm (7500
mg/m3) toluene increased ambulatory distance while the concentrations ≥ 8000 ppm (30,000
mg/m3) induced temporally biphasic effects of initial increases in activity followed by
hypoactivity. There were evident differences between groups in absolute locomotor activity
levels. The repeated exposure revealed sensitization developed in locomotor activities that was
significantly higher in each group, and that there were time course changes. These differences
in acute sensitivity and the differential shifts in sensitivity after repeated exposures among the
mouse strains suggest a genetic basis for the behavioral effects to toluene.
Demir et al. (2017) studied the effects of acute toluene toxicity on different regions of the rabbit
brain. Ten male New Zealand rabbits (healthy, average age 24 months) were given a single
dose of 876 mg/kg toluene by intraperitoneal (IP) injection, with another 10 rabbits serving as
the control group. Five hours after the injection, the blood samples were collected and the
animals were euthanized. The brain tissues were removed and processed for biochemical,
histopathological and immunohistochemical examinations. In the toluene-administered rabbits,
areas of focal vacuolar degeneration, gliosis, perivascular demyelination, and many pyknotic
cells and necrosis were detected in the brain cortex. Compared to the control group, a
remarkable excessive expansion of the blood vessels, severe degeneration of the
compensation in the cells, and almost dispersed cell borders were observed. Abnormal
malformations of the nuclei structure of the oligodendrocyte cells and structural damage of the
bodies of the sequential neurons of the hippocampus were seen as well. The authors concluded
that biochemical and histopathological methods facilitated the observation of the main toluene
exposure brain damage effects of astrocyte activation and gliosis. High levels of proapoptotic
proteins in the different regions of rabbit brain showed that toluene may trigger apoptosis in
about 3 hours.
Oshiro et al. (2011) studied the comparative abilities of the C × t and brain toluene
concentration (Br[tol]) dose metrics to accurately predict behavioral effects of the solvent in rats
upon exposures to toluene lasting up to 24 hours. This study was performed in order to
determine if Haber’s rule was applicable to toluene neurobehavioral effects. Four key
experiments were conducted involving: 1) the evaluation of the ability of a previously published
physiologically-based toxicokinetic (PBTK) model to predict Br[tol] in rats performing a lever-
pressing task (LPT) during toluene exposure; 2) use of rat telemetry data to refine the PBTK
model; 3) comparison of the C × t and Br[tol] dose metrics as they related to rat performance in
visual signal detection tasks (SDTs) during and shortly after toluene exposure for ≥21 hours;
and 4) examination of the ability to quantitatively extrapolate 1-hour exposure patterns to longer
(≥21-hour) exposures. Details of these four experiments are listed in Table 2 below. Male Long-
Evans rats (age ~ 12 weeks; 350 ± 10 g bodyweight) were used. The results suggested that
Br[tol] appeared to be a more accurate dose metric than C × t in predicting neurological effects
of exposures ≤24 hours. However, the authors also noted that behavioral tolerance occurred
after 24-hours of toluene exposure, suggesting a potential confounder in extrapolating these
effects from a 24-hour exposure to a one-hour exposure.
Table 2. Summary of acute animal studies that have a NOAEL and/or LOAEL.
Exposure duration
and Concentration Experimental animal / Effects NOAEL LOAEL
Study (ppm) (endpoints) (ppm) (ppm)
Pryor et al. 0 or 1000 ppm for 4 Rats / Hearing loss as measured 1000 and 2000 ppm,
(1984) hrs; 0 or 2000 ppm by the Conditioned Avoidance 2000 ppm 8 hrs/day
for 2 hrs; 2000 ppm Response Task single for 3 days
for 8 hrs/day for 3 exposure
days
Kishi et al. 125, 250, or 500 Rats / decreased conditioned NA 125
(1988) ppm for up to 3 hrs avoidance response
by inhalation
Exposure duration
and Concentration Experimental animal / Effects NOAEL LOAEL
Study (ppm) (endpoints) (ppm) (ppm)
Rogers et al. 1600 ppm for 6 hrs, Rats / neurobehavioral NA 1600 ppm
(1999) then 17 days of sensitization as measured by with 10
clean air, followed lever press operant testing ppm trigger
by 10 ppm “trigger” dose
exposures
Funada et al. 0 (control), 350, 5 groups of ICR mice / toluene 350 700
(2002) 700, 2500, or 3200 inhalation-related place
ppm for 20 min preference
Bowen et al. 0, 100, 2000, 8000, 5 groups of mice / increased 100 2000
(2010) and 10,000 ppm for ambulatory distance
30 min
Abbreviations: NA – not applicable
Table 2. Summary of acute animal studies that have a NOAEL and/or LOAEL (continued).
Exposure duration and Experimental animal / Effects NOAEL LOAEL
Study Concentration (ppm) (endpoints) (ppm) (ppm)
Oshiro et al. 775 or 1225 ppm ± 10 40 LPT-trained rats, age = 7.5 - NA NA

(2011) ppm for 1 hr (Groups 1 8.5 months, divided into eight
and 2), 6 hrs (Groups 3 separate groups (n = 5/group)
Experiment 1
and 4) or 24 hrs (Groups
PBPK model over-estimated
5 and 6) with necropsy
Br[tol] values in groups (5-8)
immediately post
exposed to toluene for longer
exposure or toluene
periods.
exposure as above for
24 hrs (Groups 7 and 8)
+ fresh air exposure for
6 hours prior to necropsy
Experiment 2 0 (fresh air only) or 1125 14 of 32 LPT-trained rats (age = NA NA

± 10 ppm for exposures 4-5 months) implanted with radio
lasting 6 or 24 hours, telemeters and exposed and
with necropsies tested similar to Experiment 1
immediately following, or
Consistently elevated (~10%
after a 6-hour post-
higher) heart rates in toluene-
exposure period in a
exposed rat groups versus
fresh-air environment
controls. >4-fold increases in the
PROD activity of the two groups
exposed to toluene for 24 hours
versus the control.
Abbreviations: Br[tol] – brain toluene concentration; C – concentration; LPT - lever-pressing

task; NA – not applicable; PROD - pentoxyresorufin-O-dealkylase enzyme; t – time.
Table 2. Summary of acute animal studies that have a NOAEL and/or LOAEL (continued).
Exposure duration and Experimental animal / Effects NOAEL LOAEL
Study Concentration (ppm) (endpoints) (ppm) (ppm)
Oshiro et al. 0, 1125, or 1450 ppm for 16 SDT-trained rats (age = 11 – NA 1450
(2011) exposures lasting 24 13 months)
(mild effect
hours and 1660 ppm for
Experiment 3 Overall response accuracy level for a
those lasting 21 hours
decreased and latency 1 hour
increased as functions of the C exposure)
× t and Br[tol] dose metrics;
however, Br[tol] was a better
predictor of behavior. Data used
to estimate a “mild effect level”
for a 1 hour exposure (i.e., a
doubling of the control response
latency)
Experiment 4 Same as Experiment 3 Animal data from Experiment 3 NA NA

compared to that from the 1-hr
exposure study by Busnell et al.
(2007)
1-hr data overestimated
accuracy and latency effects of
the 24-hour exposures
regardless of the dose metric
used
Abbreviations: Br[tol] – brain toluene concentration; C – concentration; NA – not applicable;
SDT – signal detection task; t – time.
6. Chronic Toxicity of Toluene

Chronic Toxicity to Adult Humans
The substantial body of studies examining the subchronic and chronic effects of toluene in
occupationally-exposed humans indicate a relationship between neurological effects and long-
term occupational exposures to toluene (e.g. ≥ 20 ppm (75 mg/m3)). The weight of evidence
from these studies indicates that various neurological effects (i.e., impaired color vision,
impaired hearing, decreased performance in neurobehavioral analysis, changes in motor and
sensory nerve conduction velocity, headache, and dizziness) are the most sensitive endpoints.
Chronic inhalation exposure of humans to toluene has also resulted in irritation of the upper
respiratory tract and eyes, sore throat, and difficulty with sleep (USEPA, 2005).
Abuse Toxicity
CNS depression and other severe neurological symptoms have been reported in chronic
abusers exposed to high levels of toluene, resulting in progressive and irreversible changes in
brain structure and function (Spencer and Schaumburg, 1985). However, exposure to other
solvents in these types of studies cannot be discounted. Early neurobehavioral changes include
anxiety, irritability, mood swings, and forgetfulness. Further exposure causes nystagmus,
slurring of speech, bilateral hearing impairment, titubation (head tremor and disequilibrium upon
standing), and a wide-based ataxic gait. A number of studies found permanent changes in brain
structure (loss of grey and white matter differentiation; cerebral, cerebellar and brainstem
atrophy) which correlated with brain dysfunction as measured by magnetic resonance imaging
(MRI), and brainstem auditory evoked response (BAER) evaluations have also been observed
in chronic toluene abusers (Rosenberg et al., 1988a; Rosenberg et al., 1988b; Filley et al.,
1990; Ikeda and Tsukagoshi, 1990; Yamanouchi et al., 1995; Caldemeyer et al., 1996).
For example, Filley et al. (1990) studied 14 chronic toluene abusers using MRI and
neuropsychological evaluations. Duration of abuse was from 24 to 252 months, with a mean of
105 months. The clinical assessment of overall neuropsychological functioning, was composed
of 12 neuropsychological tests: expanded Halstead-Reitan Battery (HRB), Wechsler Adult
Intelligence Scale (WAIS), modified Reitan’s Story Memory Test, visual reproduction component
of the Wechsler Memory Scale, the Boston Naming Test, word discrimination and complex
material tests from the Boston Diagnostic Aphasia Examination, the Thurstone Word Fluency
Test, the Peabody Individual Achievement Test, the Digit Vigilance Test, the Wisconsin Card
Sorting Test, the Tonal Memory Test, and the Grooved Pegboard and Steadiness Tests. The
assessment results indicated that three patients functioned normally, three were in a borderline
range, and eight were impaired. Independent analyses of white matter changes on MRI
demonstrated that the degree of white matter abnormality was strongly correlated (p < 0.01)
with neuropsychological impairment. The authors concluded that dementia in toluene abuse
appears to be related to the severity of cerebral white matter involvement.
Mild effects on the kidneys and liver have also been reported in solvent abusers chronically
exposed to toluene vapor, but are confounded by probable exposure to multiple solvents (NTP,
1990).
Community Epidemiological Studies

Several studies examined the health impact of ambient exposures to toluene below the U.S.
EPA RfC of 5.0 mg/m3 (1.3 ppm), particularly among vulnerable subpopulations such as
children and the elderly. However, these studies are examinations on ambient mixtures of air
toxics and therefore are less likely to provide data for developing RELs.
Toluene is one of the most frequently identified indoor residential chemical risk factors for
asthma and allergy in infants and children (Mendell, 2006). However, there is a lack of evidence
that toluene on its own acts as a sensitizing agent that leads to asthma in children. More likely,
co-exposures to other antigenic materials (e.g. dust mite antigen) can lead to asthma, and
toluene is simply acting as a marker for another correlated compound or substance.
Rumchev et al. (2004) surveyed 88 children aged 6 months to 3 years in Australia who were
diagnosed with asthma, together with 104 children of the same age group without an asthma
diagnosis (controls). Questionnaire information collected included the health status of the
children, exposure to VOCs including toluene, average temperature and relative humidity in the
living room of each participating family. The median concentration of indoor toluene was 17.1
(range 0.01 – 153.9) µg/m3 (4.54 ppb, 0.0027 – 40.84 ppb). The result showed that the children
with asthma were exposed to significantly higher VOCs than controls (p < 0.01), and toluene
provided the third highest odds ratio for asthma. The risk of asthma increased two-fold for every
10 µg/m3 (2.7 ppb) increase in the indoor concentration of toluene. The authors concluded that
indoor exposure to VOCs, including toluene, at levels lower than current recommended value
may still increase the risk of childhood asthma.
Delfino et al. (2003a and 2003b) investigated the correlation between asthma symptoms in
children and ambient air VOCs including toluene. Twenty-one Hispanic children with mild
asthma from a Los Angeles community with high VOC levels provided symptom diaries and
peak expiratory flow (PEF) data daily for three months, and their exhaled VOC samples were
analyzed by GC-MS. Toluene was shown to have a positive association with asthma symptoms.
Hulin et al. (2010) compared the asthmatic effects of indoor air pollutants in urban homes with
those in rural houses, involving two populations of children living in a city (32 asthmatics and 31
controls) and in the countryside (24 asthmatics and 27 controls). The pollutants, including
toluene, were assessed at the homes for 1 week. The urban homes were shown to have up to 2
times higher pollutant levels than rural homes. In both populations, toluene was significantly
related to a higher risk of asthma.
In the study of Bentayeb et al. (2013), 567 buildings in Metropolitan areas of France were
randomly selected and 1,012 inhabitants over 15 years of age, including 144 individuals over
age 65, were surveyed for breathlessness and chronic bronchitis, together with the indoor
concentration of aldehydes and 20 VOCs in their dwelling. While similar levels of indoor air
pollutants were found for the elderly and other inhabitants, increased toluene concentrations
were significantly associated with breathlessness and chronic bronchitis in the elderly but not in
the rest of the population. Adjusted odds ratios (95% confidence interval) of 3.36 (1.13, 9.98)
were observed in the elderly, in comparison with 0.91 (0.59, 1.39) in the other inhabitants.
Xu et al. (2009) found that blood toluene levels were correlated with increased odds of
cardiovascular disease (CVD). The authors used the 1999–2004 National Health and Nutrition
Examination Survey (NHANES) data to examine the relationship between alkylbenzene levels
(toluene, styrene, ethylbenzene, and the xylenes) and CVD prevalence. Levels of all five
alkylbenzenes demonstrated linear dose-response trends. For toluene, 389 subjects had an
average blood concentration of 0.751 ng/mL, the odds ratio was 2.30 (50th-85th percentiles) and
3.49 (≥85th percentiles). Further studies are needed to explore associations between these
highly prevalent pollutants and CVD.
Occupational Studies
Neurological effects (CNS, sensory irritations, neurobehavioral and psychometric
tests)
Wilson (1943) reported that occupational exposure of workers to concentrations of commercial

toluene ranging from 50 to 200 ppm (200 to 750 mg/m³) for periods of 1 to 3 weeks resulted in
headaches, lassitude, and loss of appetite. At 200 to 500 ppm (750 to 2000 mg/m³), symptoms
of nausea, bad taste in the mouth, slightly impaired coordination and reaction time, and
temporary memory loss were also observed. Exposure to 500 to 1500 ppm (2000 to 5600
mg/m³) resulted in palpitations, extreme weakness, pronounced loss of coordination, and
impaired reaction time. Red blood cell counts were decreased and there were 2 cases of
aplastic anemia. Commercial toluene likely contained significant levels of benzene, which may
have caused the red blood cell effects.
Matsushita et al. (1975) evaluated the clinical and subjective responses of industrial workers
exposed to toluene vapor. Subjects included 38 female shoemakers occupationally exposed to
toluene (and slight amounts of gasoline) and a control group of 16 female workers from the
same manufacturing plant who had never been exposed to any solvent. The mean duration of
solvent exposure was 3 years and 4 months, and the mean age of the exposed subjects was
20.7 ± 5.2 years, which was similar for the control group. Toluene concentrations in air in the
workroom of the exposed group ranged between 56 to 380 mg/m3 (15 and 100 ppm) in
February and September, with an average of 226 to 263 mg/m3 (60 to 70 ppm), and between 38
and 750 mg/m3 (10 and 200 ppm) with an average of 100 ppm in August of the study period. A
health questionnaire was administered to collect subjective symptoms, and clinical findings were
drawn from urinalysis and tests for liver and neuromuscular function.
Subjective symptoms of the exposed subjects were significantly different from the controls with
respect to dysmenorrhea (p < 0.05) and feeling uneasy about solvent vapor (p < 0.01); exposed
subjects also reported a sensation of general weakness and itching and dermatitis of hands, but
these did not reach statistical significance by ꭓ2-test (p < 0.1). The only significant hematological
difference between groups was a marked increase in the appearance rate of Mommsen’s toxic
granules in peripheral neutrophils in the exposed group (7.6 ± 5.6%, 13/38 subjects; p < 0.05)
relative to controls (3.8 ± 3.4%, 1/16 subjects). Excretion of hippuric acid in urine was
significantly elevated in the exposed group (3.26 ± 0.82 mg/ml) compared to controls (0.36 ±
0.24 mg/ml). Neuromuscular function tests revealed a significant difference between the
exposed and control groups with abnormal tendon reflex of the patella (p < 0.05); other
differences reported (abnormal tendon reflex of the ankle) were not statistically significant by
application of t- and ꭓ2-tests (p < 0.1). No significant correlation was found between the duration
of toluene vapor exposure and the appearance rate of abnormal findings.
Up to 101 solvent workers exposed to toluene in shoe-making factories were examined for
subjective symptoms, and changes in hematology and serum and urine biochemistry (Yin et al.,
1987). The mean TWA toluene exposure of the subjects was 42.8 ppm (161 mg/m3) and the
average exposure duration was 6.8 years. Concurrent exposure to benzene (TWA 1.3 ppm)
also occurred in these workers. Compared to a group of 127 control workers, the prevalence of
subjective symptoms was greater (p < 0.01) in the toluene-exposed workers during work and in
the past 6 months. The most common symptoms were sore throat, dizziness, and headache.
Nose and/or eye irritation during work and difficulty in sleeping were also reported. When the
toluene-exposed workers were separated into a low exposure group (< 40 ppm, 151 mg/m3) and
a high exposure group (≥ 40 ppm, 151 mg/m3), the prevalence of the three most common
symptoms appeared to be dose-related. Their hematology was essentially normal and serum
and urine biochemistry was unremarkable.
Orbaek and Nise (1989) examined the neurological effects of toluene on 30 rotogravure
printers, 33-61 years of age (mean 50), employed at two Swedish printing shops for 4-43 years
(median 29) in 1985. Mean toluene exposure levels at the two printing shops were 43 mg/m3
(11.4 ppm) and 157 mg/m3 (41.8 ppm); however, before 1980 the mean exposure levels had
exceeded 300 mg/m3 (79.8 ppm) in both shops. The authors noted that rotogravure printing
provides an occupational setting with toluene exposure not confounded by exposures to other
solvents. Comparisons were made to a reference group of 72 men aged 27-69 yrs (mean 47
yrs). The alcohol consumption of both the workers and referents was also determined (< 200
g/week or > 200 g/week). Neurological function in the workers and referents was evaluated
using interviews and psychometric testing; the results from each of the two printing shops were
pooled. The printers reported statistically significantly (p < 0.05) higher occurrences of fatigue
(60%), recent short-term memory problems (60%), concentration difficulties (40%), mood lability
(27%), and other neurasthenic symptoms. The printers also scored significantly worse than
referents in a number of psychometric tests, including synonym, Benton revised visual retention,
and digit symbol tests, even after adjustment for age. For all comparisons, tests for interaction
between the effects of toluene exposure and alcohol consumption were not statistically
significant.
A battery of neurobehavioral tests was performed in 30 female workers exposed to toluene

vapors in an electronic assembly plant (Foo et al., 1990). The average number of years worked
was 5.7 ± 3.2 (mean ± standard deviation (SD)) for the exposed group and 2.5 ± 2.7 years for a
control group. Study subjects did not smoke tobacco or drink alcohol, were not taking any
medications, and had no prior history of central or peripheral nervous system illness or
psychiatric disorders. The exposed group of workers inhaled a time-weighted average (TWA) of
88 ppm (330 mg/m3) toluene while the control workers inhaled 13 ppm (49 mg/m3). The control
group, matched for age, sex and ethnicity, was selected from another section of the facility
where toluene was not used, but had low levels of toluene due to cross contamination of work
areas. Post-shift blood toluene concentrations were also determined in the workers. Mean blood
toluene levels were 1.25 mg/l in the exposed group and 0.16 mg/l in the control group. In a
previous study of factory workers at the same plant, Foo et al. (1988) observed a linear
correlation between blood toluene concentration and TWA exposure.
Foo et al. (1990) conducted the neurobehavioral tests during the work week on Wednesdays
and Thursdays prior to start of the work shift. A statistically significant decrease in
neurobehavioral performance was observed in the exposed workers for 6 out of 8 tests (p <
0.05, Student’s t test). Analysis of covariance showed that the performance in each of these six
positive tests was related to the TWA concentration of toluene. However, no statistically
significant relationship was found between duration of exposure and performance in the
neurobehavioral tests. Decreased performance of exposed workers were observed for the
grooved peg board test that measures manual dexterity, the trail making, visual reproduction,
Benton visual retention, and digit symbol tests that measure visual scanning ability, and the digit
span test that measures verbal memory. Simple reaction time and finger tapping tests, which
measure motor speed, were not affected by toluene exposure. The authors noted that the lack
of effects on motor speed, while other neurobehavioral tests of toluene exposure exhibited
decreased performance, matches the results of the acute toluene exposure study by Echeverria
et al. (1989).
Irritant effects of toluene in the workers were not discussed by Foo et al. (1990), although the
study states that no clinical symptoms or signs were observed. This finding suggests that the
neurobehavioral effects observed were subclinical. Potential concurrent exposures to other
chemicals were not specifically addressed. In this study, 88 ppm was considered a LOAEL for
central nervous system effects. However, the workers designated by the authors to be controls
did not comprise a true control group, since they were exposed to an average of 13 ppm (49
mg/m3) toluene. This may have resulted in an underestimation of the effects of exposure to 88
ppm (330 mg/m3) toluene, in particular because the authors noted a dose-response relationship
between TWA toluene air concentration and neurobehavioral performance in most of the tests.
Murata et al. (1993) investigated peripheral neurotoxicity in toluene-exposed workers. Subjects

included 10 male workers who were between the ages of 20 and 77 years (mean age 39 years)
and exposed to toluene at a rotogravure printing company in Saitama, Japan for 1-36 years
(mean 11 years) at an estimated exposure level of 41-224 ppm (mean 83 ppm) toluene. These
men ingested alcohol (0-480 ml/week, mean 88), smoked tobacco (0-30 cigarettes/day, mean
7), and had no history of cardiovascular or neurologic disease or alcohol dependency. Control
subjects were 10 healthy, unexposed men of similar economic status who were not significantly
different in age, skin temperature, or alcohol and tobacco consumption from the exposed
workers. Blood and urine samples were collected for analyses of toluene and hippuric acid
levels, respectively. Electrophysiological tests were conducted to measure cardiac autonomic
function as follows: coefficient of variation in electrocardiographic R-R intervals (CVRR),
distribution of nerve conduction velocities (DCV), maximal motor and sensory nerve conduction
velocities (MCV and SCV) in the median nerve, and the high-frequency (HF) and low-frequency
(LF) components of the CVRR corresponding with parasympathetic (C-CVHF) and sympathetic
(C-CVLF) activities, respectively.
Blood toluene concentrations in the exposed workers averaged 526 µg/L (range, 147 to 1,119)
and urinary hippuric acid levels averaged 1.61 g/L (range, 0.94 to 3.9). Blood toluene and
urinary hippuric acid concentrations were not provided for the control group. Relative to
unexposed controls, the CVRR and C-CVHF were significantly lower in the exposed workers (p <
0.05), suggesting reduced nerve conduction velocities. The other significant finding was a
reduction of the MCV in the forearm and the SCV in the palm in the exposed workers compared
with controls, with observed matched differences of -3.7 ± 1.9 m/sec (p < 0.01) and -4.6 ± 4.4
m/sec (p < 0.05), respectively. Heart rate did not differ between exposed subjects and controls,
and no significant association was found for electrophysiological data and blood toluene or
urinary hippuric acid levels, or duration of toluene exposure among exposed subjects.
Vrca et al. (1995) studied the effects of toluene on visual evoked potentials (VEPs), which are
measurements of electrical signals produced in the brain in response to light stimuli. The
exposed group included 46 male and 3 female employees (total n = 49) of a printing-press
factory in which toluene was used exclusively as an organic solvent for 30 years prior to the
study. Average airborne toluene concentrations ranged from 226 to 301 mg/m3 (60 to 80 ppm)
in the printing-press factory; average employee age was 42.3 ± 6.8 years (range = 24 - 55
years); and average employment length was 21.4 ± 7.4 years (range = 4 - 30). The control
group (n = 59; 54 males; 5 females) included factory workers producing rotating coils for electric
motors who were not occupationally exposed to any neurotoxic substances known to the study
authors. Average age was 43 ± 7.2 years (range = 23 - 55 years); and average employment
length was 20.6 ± 7.7 years (range = 4 - 32 years).
Subject VEPs were measured with a brain imager (electroencephalograph; EEG) with
electrodes placed at the occiput (the location of the visual cortex) for recording VEPs. According
to the authors, N75, P100, and N145 waves of VEPs were analyzed by summing up the same
values for the right and left eye in each subject and dividing by two. Measured brain wave
parameters included the time of onset, attainment of maximal amplitude (wave latency), offset,
and total duration (offset minus onset latency), as increases in latency and decreases in
amplitude could indicate pathology/dysfunction.
Toluene exposure was determined using the peripheral blood concentration of toluene, and the
urinary creatinine-adjusted concentrations of hippuric acid and ortho-cresol in 36 exposed and
27 unexposed workers (sex ratios not stated). The results suggested that pre-work-shift
concentrations of toluene in blood were significantly greater (p < 0.00096) in the exposed
workers relative to their unexposed counterparts, as were the pre (p < 0.05) and post (p <
0.001) work-shift hippuric acid levels in urine. Toluene-exposed workers exhibited significantly
(p ≤ 0.01) higher wave (P100, N45, and N175) amplitudes and significantly longer P100 latency
(p < 0.05) than unexposed workers. Though the authors stated the former may be due to more
excitable wave generators in the visual pathway, they also recognized that P100 latency was a
more sensitive toluene-induced VEP parameter, and suggested the increased latency may be
due to reduced wave conductivity in certain segments of the visual pathway.
Using the same subjects from their 1995 study, Vrca et al. (1996) evaluated the effects of
occupational toluene exposure (60-80 ppm) on brainstem auditory evoked potentials (BAEPs).
As above, workers were asked to refrain from taking medicine for 5 days prior to testing. BAEPs
were measured on a Monday, prior to the start of the work-shift, by subjecting each ear to
testing. Subjects were tested in a dark, temperature-controlled (range = 19 - 21°C) room, and
asked to keep their eyes closed. While one (test) ear was stimulated with a click stimulus [2048
times, 15 stimuli/second at 70 decibel sound pressure level (dB SPL)], the other (unstimulated
ear) was simultaneously subjected to masked noise (50 dB SPL). Brain responses occurring
within the first millisecond after commencement of stimulation were recorded by EEG. Waves
P1 - P5 were analyzed by amplitude, latency, and interpeak latency. The wave forms had
abscissae (horizontal, x-axis measures) and ordinates (vertical, y-axis measures). The values
for the left and right ear in each subject were summed and divided by 2, and data were
statistically analyzed as above.
Results indicated that all wave latencies were significantly longer (p ≤ 0.01) and amplitudes
were significantly smaller (p ≤ 005) in exposed versus unexposed workers. According to the
authors, smaller wave amplitudes suggested changes to the whole length of the auditory
pathway, through the brainstem. Significantly (p ≤ 0.01) longer inter-peak latencies were also
observed for exposed workers when compared to their unexposed counterparts. These
latencies included P3 – P4, P1 – P5, P3 – P5, and P1 – P4. Given the interpeak latencies and
that of the P1 wave, specifically, the authors suggested that the sites most affected by toluene
may be the extramedullary part of the auditory pathway (the Cortti organ and auditory nerve)
and the high medullary part up to the level of the auditory colliculus, with the intervening section
of the pathway more resistant to the effects of toluene.
A group of 49 printing-press workers occupationally exposed to toluene for approximately 21.6

years was studied by Vrca et al. (1997a). Toluene exposure levels were determined from blood
toluene and urinary hippuric acid levels, and were estimated to range from 40-60 ppm (151-226
mg/m3). No control group was used. Brain-evoked auditory potential (BEAP; similar to BAER)
and visual evoked potential (VEP) measurements were performed on a Monday morning after a
nonworking weekend. There was a significant increase in the latencies of all the BEAP waves
examined (except for P2 waves), as well as in the interpeak latency (IPL) P3-P4, while IPL P4-
P5 decreased significantly with the length of exposure. No correlation was noted between the
amplitude of BEAP waves and the length of exposure. The amplitude but not the latency of all
the VEPs examined decreased significantly with the length of exposure.
The Vrca et al. (1997b) report expanded upon the Vrca et al. 1995 publication by reporting the
effects of toluene on the P300 wave. This wave is not like the P100 wave, which is generated by
an external stimulus, but rather is generated by a person’s reaction to a stimulus as part of a
decision-making process. For example, to measure the P100 waves, VEPs were provoked
using alternating checkerboard patterns on a computer screen. To measure P300 waves,
subjects are presented with a sequence of events such that each event can be categorized as
“frequently” or “infrequently” occurring. In the 1997 study by Vrca et al., the alternating
checkerboard pattern fell into the former category. A separate “rare” stimulus generated the
P300 wave about 300 ms after stimulus onset. As P300 is involved with the process of memory
modification or learning, and stimuli appear to be learned if, and only if, they are novel. The
P300 wave is smaller in subjects with decreased cognitive ability relative to age-matched
normal subjects. Results from this study suggested that toluene caused delayed VEPs
(increased latency) during mental engagement (i.e. presentation of the “rare” stimulus, which
may reflect adverse changes to intracranial structures like the brainstem and hypothalamus.
Boey et al. (1997) examined 29 electronic assembly plant workers chronically exposed to
toluene (4.9 ± 3.5 years) for neurobehavioral deficits on a midweek morning (i.e., more than 12
hours but less than 24 hours after exposure ceased). The exposed workers had an 8-hour TWA
toluene exposure of 90.9 ppm (343 mg/m3) and were compared to a matched control group of
29 workers from the same electronics factory that had a low level of toluene exposure (8-hour
TWA 12.2 ppm, 46.0 mg/m3). Neurobehavioral effects were investigated using the logical
memory, digit span, visual reproduction, trail making, symbol digit modality, and grooved
pegboard tests. Significant decrements were observed in 9 of 14 examiner-administered tests of
exposed workers compared to referents analyzed by ANOVA with years of education as a
covariate.
Eller et al. (1999) evaluated the chronic effects of toluene exposure on the central nervous
system of workers in a rotogravure plant. Ninety-eight male workers from a selection pool of 107
(92%) underwent neuropsychological examination using a Cognitive Function Scanner, and
neurological examination by computerized methods measuring coordination ability, tremor and
position stability. In addition, measures of symptoms and former exposure were obtained by
questionnaire. The workers were divided into three groups: Group 0 with no exposure to organic
solvents (n = 19); Group 1 with exposure to TWA <20 ppm (75 mg/m3) of toluene for less than
13 years (n = 30, average exposure time 7.7 yr) and Group 2 with exposure for more than 12
years (n = 49, average exposure time 25.5 yr). Within Group 2, all workers had been exposed to
levels exceeding 100 ppm (380 mg/m3) for at least 4 years, with 37 of the workers (75%)
exposed for 10 or more years before 1983 at that level.
Among the findings by Eller et al. (1999), no significant differences were found between Group 0
and Group 1 regarding symptoms and the results of neuropsychological and neurological
function tests. However, Group 2 differed significantly from the other two groups for increased
symptom index score (p = 0.04), particularly with answers on the questionnaire regarding the
ability to concentrate, and reduced memory and fatigue. Group 2 scored much poorer on
neuropsychological tests compared to group 0 for visuospatial function (p = 0.06), number
learning (p = 0.04) and word recognition (p = 0.02), while only one marginal deficit (p = 0.05) in
the neurological function tests (finger tap, left hand) was observed. The authors concluded that
the exposure to toluene in Group 2 for over 12 years with an estimated TWA over 100 ppm (380
mg/m3) for at least 4 years (range: 4-27 years) was associated with impaired
neuropsychological function.
Deschamps et al. (2001) studied the cognitive function impairment from long-term toluene
exposure on 72 workers (42 men, 30 women) compared with 61 non-exposed workers. Toluene
exposure dosage from personal samplers and atmospheric sampling showed 9 to 467 ppm (34
to 1,756 mg/m3), and the mean duration of exposure was 19.9 ± 0.9 years. The results from a
subjective questionnaire and psychometric tests showed no significant differences between
exposed and non-exposed workers except that the mucosal irritation score was significantly
higher in exposed than in non-exposed workers (p < 0.005). According to the authors, several
workers exposed to high atmospheric concentrations of toluene did not show statistically
different results in the subjective questionnaire for cognitive and neurological functions or for the
psychometric tests.
Neubert et al. (2001) studied a large cohort of 1,290 rotogravure printers in Germany
occupationally exposed to toluene for an undefined number of years, for whom questionnaire on
subjective symptoms and psycho-physiological and psycho-motoric tests were assessed,
compared with 200 unexposed workers. No neurological symptoms or impairment on overall
performance was observed in the tests, except for the flicker fusion frequency test. A significant
decrease effect on visual perception was found among a subgroup of 47 workers exposed to an
estimated 70–80 ppm (263–301 mg/m3) toluene for 6 hours during a daily shift. In this study, a
LOAEL for impaired visual perception was estimated as 75 ppm (282 mg/m3).
Gericke et al. (2001) studied the effect of long-term (at least 20 years) low-concentration toluene
exposure on 1,077 male rotogravure factory workers. Toluene exposure concentrations were 24
ppm (90 mg/m3) for printers and 4.5 ppm (17 mg/m3) for non-printers (N = 109). Physical exam,
neuropsychological tests and a questionnaire of subjective symptoms showed no significant
increase in neurological symptoms or overall performance on neuropsychological or
psychomotor tests from the exposed workers compared with unexposed workers.
Chouaniere et al. (2002) examined 128 toluene-exposed printing workers (14 women and 114
men) from two plants 48 hours after their shift ended for psychometric changes using the
Neurobehavioral Evaluation System (NES) tests. Worker exposure was monitored for 3 or 4
days prior to testing to estimate exposure based on workshop, job type, and shift in the plants.
The average exposure duration was 14 ± 10 years with current TWA toluene exposures ranging
from 0 to 27 ppm (102 mg/m3), although past exposures were estimated to range from 0 to 179
ppm (675 mg/m3).
Multiple regression analysis found a statistically significant dose-effect relationship between

toluene exposure and decrements for the Digit Span Forwards (p = 0.04) and Digit Span
Backwards tests (p = 0.01) (both measures of short-term memory performance), after correction
for the confounders of sex, age, synonym score (for education), history of CNS diseases,
alcohol consumption, psycho-active drugs used in last day, concentration in performing tests
and computer experience. Neurotoxic symptoms were obtained through a questionnaire
(EUROQUEST), including 83 items within 5 categories: (1) neurological symptoms and
psychosomatic symptoms; (2) acute symptoms; (3) mood, memory, concentration, fatigue,
sleep disturbances; (4) environmental susceptibility; (5) anxiety, perception of health status and
life. The results showed that the neurotoxic symptoms were not significantly correlated with
current exposure, and no association was found between estimated cumulative exposure and
either psychometric performances or neurotoxic symptoms. Although specific NOAELs and/or
LOAELs were not determined in this study, the results indicate low current exposures to toluene
were associated with decrements of memory test performance.
Zupanic et al. (2002) investigated the possible effects of long-term occupational exposure to
toluene below 100 ppm on psychological performance and subjective symptoms. Male workers
(N=278, mean duration 14.9 years) from 14 rotogravure printing plants in Germany were
examined. A “high dose” group of 154 workers (printing area) had a mean lifetime weighted
average exposure (LWAE) of 45.1 ppm (170 mg/m3) and a mean current exposure of 24.7 ppm
(93.1 mg/m3), and a “low dose” group of 124 workers (end-processing area) had a mean LWAE
of 9.3 ppm (35 mg/m3) and a mean current exposure of 3.3 ppm (12 mg/m3). Examinations were
performed on psychomotor performance (steadiness, line tracing, aiming, tapping, and peg
board) and subjective symptoms. Analysis of variance (ANOVA) found no significant differences
between the two groups. There was no significant correlation between long-term toluene
exposures and adverse psychological performance or subjective symptoms of exposure at
levels ranging between 1 - 88 ppm.
Seeber et al. (2004) reported a study using the same data set from Zupanic et al. (2002) above,
with a subsample of 192 workers that went through all 4 repeated examinations for the cognitive
function effects of occupational exposure to toluene lower than 50 ppm (188 mg/m3). Current
exposure levels were grouped as “high” (printing area, average concentration 26 ppm (98
mg/m3)) or “low” (end-processing, average concentration 3 ppm (11 mg/m3)). Past exposure
levels (LWAEs) were grouped as “high” (45 ppm (170 mg/m3)) or “low” (9 ppm (34mg/m3)).
Exposure durations were grouped as “long-exposure” (average exposure 21 years) and “short-
exposure” (average exposure 6 years). Psychological tests included tests of attention, memory,
and psychomotor functions. The results showed neither past exposure nor current exposure
resulted in significant impacts on the psychological test performance. The author’s conclusion
was that long-term toluene exposure below 50 ppm (188 mg/m3) did not show psychological
effects on cognitive functions of the above printing workers.
Seeber et al. (2005) further analyzed the above dataset of 192 workers for 4 examinations, with
more details on sensory functions. The “high” group had 106 workers, current toluene exposure
of 26 ppm (98 mg/m3) and LWAE of 45 ppm (170 mg/m3), while the “low” group had 86 workers,
current exposure of 3 ppm (11 mg/m3) and LWAE of 9 ppm (34 mg/m3). Measured sensory
functions included vibration thresholds, color discrimination and auditory thresholds.

Psychological performance tests included attention, memory and psychomotor functions. An
odds ratio statistical analysis revealed no significant relationship between long-term toluene
exposure below 50 ppm and impaired psychological functions among the “high” exposure group
workers.
Kang et al. (2005) examined 54 workers in Korea (average 35.3 years of age, 11.7 years of
education) from three different industries for the effects of chronic exposure to toluene. The
workers were divided into three exposure groups, according to the periodic work environment
measurement of toluene concentrations: high exposure group, 20 workers, exposed for 5.4 ±
5.3 years to 70-80 ppm (263-301 mg/m3) of toluene; moderate exposure group, 13 workers,
exposed for 8.3 ± 9.5 years to 20-30 ppm (75-113 mg/m3) of toluene; low exposure group, 21
workers, exposed for 10.9 ± 6.4 years to less than 1 ppm (3.8 mg/m3) of toluene. The results of
a battery of neurobehavioral tests on these workers showed significant impairments on the
finger tapping (ANOVA, p = 0.002) and selective attention tests for the high exposure group of
workers compared with the low exposure group (p = 0.000), while the test results for the
moderate exposure group of workers did not differ significantly from the low exposure group (p
> 0.05).
Nordling Nilson et al. (2010) conducted a longitudinal follow-up study to examine effects of long-
term toluene exposure on cognitive functions later in life. Study participants, who were initially
examined and studied in 1983/1984, were re-examined 20 years later and included 12 male
rotogravure printers previously exposed to toluene and 19 male referents. The follow-up study
population was much smaller than the initial study (originally comprised of 30 male printers and
a reference group of 50 sugar refinery workers and 22 railway carriage repair shop workers),
after applying exclusion criteria of present/past solvent exposure, diagnosis of disease affecting
central nervous system, and non-age-matched referents among available (living) subjects. The
mean ages of the printers and referents were 70 ± 6.8 years (range, 59 to 80) and 67 ± 7.3
years (range, 57 to 84), respectively. Most of the subjects were retired, and the mean number of
years of education for printers and referents was 9 and 11, respectively. There was no
significant difference in cognitive performance between printers and referents at the time of the
initial study in 1983/1984.
Cumulative exposure assessments for solvents and potential neurotoxicants were conducted for
each subject based on data collected during the initial and follow-up studies. Among printers,
toluene was the primary chemical of exposure, which occurred before 1985 for an average of 35
years. Estimated retrospective toluene exposure levels were 400 ppm (1500 mg/m3) during the
1950s–1960s, after which exposures gradually declined due to preventive measures. At the two
printing plants where the printers were employed the mean toluene levels were 43 and 157
mg/m3 by the mid-1980s when their occupation as printers ended. No exposure to toluene or
other organic solvents occurred during the follow-up period; however, 4 printers were exposed
after 1992. The referent group had no significant exposure to toluene or other solvent at any
time, nor did the printers have any significant exposure to solvents during their leisure time
relative to the general population. Low-level lead exposure was reported for one printer who
sealed packages for two years in the late 1930s, and for 3 referents while plumbing up to 1968
and using paint in the mid-1940s.
Each subject underwent extensive clinical interviews and medical examinations that focused on
signs of neurological or psychiatric impairment and alcohol and drug use. Physical examination
and standard screening blood tests were included. No disease or medical discrepancy relevant
to cognitive function was found. No alcohol overuse was reported, and there were 4 smokers
and 6 ex-smokers among the printers and 2 smokers and 10 ex-smokers among the referents.
Study participants were administered questionnaires regarding social interactions and
subjective cognitive impairments, as well as the same neuropsychological tests used in the
initial study and additional tests sensitive to the influence of aging.
There was a decline over time observed on test performance from the initial study to follow-up in
all except one test (reasoning) for both printers and referents. However, the decline in reasoning
was evident only in the printers while performance of the referents remained stable. A dose-
effect relationship was evident in lower performance with increasing cumulative toluene
exposure for reasoning [F (1, 8) = 6.99, p = 0.03]. Additionally, the decline in associative
learning over time was more notable in printers than in referents, and a significant interaction
effect of time and exposure group was found (p < 0.05, analysis of variance (ANOVA)).
Performance of printers was significantly worse than referents in sustained attention (p < 0.05)
and verbal memory related to categories involved in cued recollection of organizable words (p =
0.05). Also noteworthy were an observed decline in attention and memory in 75% of printers vs.
25% of referents, and a somewhat higher rating for depression among printers relative to
referents (p = 0.05). Printers did not indicate more subjective cognitive impairments than
referents, nor were there significant differences between the two groups regarding ratings for
social interactions.
Color vision impairment

The physiology of color perception and color vision abnormalities were reviewed by Iregen et al.
(2002). Cones, one of the two main types of visual receptor cells in the human eye, are
responsible for the perception of color. Among the cone cells, short wavelengths (i.e., the color
blue) are perceived by S cones, which represent less than 10% of the total cone population. S
cones are believed to be more sensitive to diseases of the eyes and exposure to various drugs
and chemicals. Thus, acquired color vision defects regarding blue-yellow dimensions are often
reported. Color discrimination abilities seem to be especially sensitive to impairment following
exposure to industrial chemicals including toluene (Geller and Hudnell, 1997). Occupation-
related color vision impairment usually results in blue-yellow color discrimination loss (Type III
dyschromatopsia) or, less frequently, a combination of blue-yellow and red-green loss (Type II
dyschromatopsia), while congenital dyschromatopsias more frequently result in red-green
deficits (Type I dyschromatopsia) (Gobba and Cavalleri, 2003).
However, the pathogenesis of occupational color vision loss remains unclear. It is probably a
result of damage to the optic nerve rather than damage to ocular structures (Zavalic et al.
1998c). It has also been proposed that the color loss is due to a direct action of neurotoxins on
receptors, possibly on the cone’s membrane metabolism, and/or to an interference with
neurotransmitters within the retina (Gobba and Cavalleri, 2003). As a generalized mechanism of
action, the liposoluble nature of toluene might play an important role in affecting the visual
system, such as visual pathway degeneration, direct impact on photoreceptor function, and
cortical or retinal changes in the neurotransmitter system (Costa et al. 2012). Additionally, color
vision impairment may involve toluene interference with dopaminergic mechanisms of retinal
cells or demyelinization of optic nerve fibers, which have a high lipid content (Zavalic et al.,
1998b; Mutti and Franchini, 1987). Finally, lipophilic solvents such as toluene can alter
membrane permeability, which involves intercalation of toluene into the lipid bilayer of nerve
membranes producing an anesthetic action (Bowen et al., 2006; Evans and Balster, 1991).
However, membrane alteration appears to occur at concentrations higher than those that result
in neuronal excitability and neurotoxicity.
Studies have found that color vision impairment progressed with increasing cumulative
exposure to toluene and other neurotoxic chemicals. However, whether color vision impairment
due to toluene exposure is reversible or long-lasting beyond a long weekend of non-exposure is
not clear. Studies have been conducted with other solvents that have examined the reversibility
of this effect. Styrene is one of the most investigated compounds with regard to color vision
loss. In their review, Gobba and Cavalleri (Gobba and Cavalleri, 2003) noted that the cumulative
data on the reversibility of color vision loss following chronic styrene exposure is conflicting. For
example, a one-month interruption in exposure to styrene did not improve color vision in one
study, while another study observed complete recovery. In studies that followed a reduction in
styrene exposure of 1 year or more, no improvement was found in one study while another did
see an improvement. Gobba and Cavalleri (2003) suggested longer exposure duration and
higher exposure concentration may be a factor leading to long-lasting color vision loss following
cessation of exposure.
Nakatsuka et al. (1992) examined color vision loss in two groups of workers: one group of 261
workers with previous occupational exposure to solvents and a second group of 120 (48 men
and 72 women) non-exposed control workers. Among the solvent workers, 63 men and 111
women were exposed to predominantly toluene (46 ppm (170 mg/m3) as the geometric mean
concentration); the rest were exposed to either tetrachloroethylene alone (13 ppm, 49 mg/m3),
or a mixture of tetrachloroethylene (12 ppm, 45 mg/m3) and trichloroethylene (7 ppm, 26
mg/m3). The exposure duration history of the workers was not provided. Color vision loss was
first screened using Lanthony's new color test, and then confirmed by Ishihara's color vision
test. The only instances of color vision loss that were detected in either the exposed workers or
the controls were six cases of red-green loss (all in men), which was probably congenital in
nature rather than acquired through workplace exposure. Further examination for distribution of
red-green loss cases among men showed no correlation to toluene exposure. Additionally, no
gender-related differences in color vision loss were observed.
Zavalic et al. (1996) examined color vision in 41 female shoe workers exposed to toluene and
29 non-exposed female workers as controls. For the exposed group, average toluene air
concentration throughout the working week was 35 ppm (132 mg/m3) and average toluene
concentration in blood on Wednesday before work was 0.36 ppm (1.33 mg/m3), average
exposure duration (years of work) was 19.90 ± 7.10 years. Color vision was tested through the
Lanthony-D-15 desaturated panel in standard conditions. Since alcohol can be a major
confounding factor in acquired color vision impairment, the color confusion index (CCI) was
corrected for alcohol intake with a formula residual CCI (RCCI) = CCI – (1.129 + 0.000595 ×
alcohol intake). The results showed that both CCI and RCCI values were significantly higher in
the exposed group than in the non-exposed (p < 0.05).
Valic et al. (1997) studied acquired color vision impairment (dyschromatopsia) in 138 persons
who were exposed to solvents (toluene, xylene, trichloroethylene and tetrachloroethylene)
combined with alcohol consumption, against 100 nonexposed controls. The gender of the
subjects was not provided. Of those subjects, 105 had toluene or xylene exposure. Color vision
loss was tested with Lanthony’s D-15 method and assessed based on Bowman’s color
confusion index (CCI). The results showed that more than 250 g per week alcohol consumption
combined with solvent exposure significantly elevated CCI (p = 0.0044).
Zavalic et al. (1998a, 1998c) investigated color vision impairment in three groups of workers,
two groups occupationally exposed to toluene and a control group. The first exposed group (E1)
of 41 workers (initially 45 women and 3 men, with7 subsequently excluded due to medical
issues or insufficient exposure duration) was exposed to a geometric mean toluene air
concentration of 35 ppm (132 mg/m3) (range 11.3–49.3 ppm (42.6–186 mg/m3)) for an average
of 16.21 ± 6.10 yr and the second exposed group (E2) of 32 subjects (initially 36 men and 3
women, with7 subsequently excluded) was exposed to a geometric mean toluene air
concentration of 156 ppm (590 mg/m3) (range 66.0–250.0 ppm (250–940 mg/m3)) for an
average of 18.34 ± 6.03 yr. Color vision impairment is dependent on age and alcohol
consumption, so these factors were controlled for in the color vision impairment study. The
nonexposed group (NE) comprised 83 subjects (initially 61 men and 31 women, with 9
subsequently excluded). Color vision was evaluated by the Lanthony D-15 desaturated test
according to Verriest's classification: type I, loss in the red-green range; type II, loss in the blue-
yellow and red-green ranges, and type III, loss in the blue-yellow range. Subjects were
classified as dyschromates if specific acquired loss was determined in at least one eye. In both
exposed groups, exposure was evaluated by measurement of the concentration of toluene in
the ambient air and in the blood. In group E2, levels of hippuric acid and orthocresol in urine
after the work shift were also determined.
The prevalences of the total dyschromatopsia (type III + type II) in the three groups of subjects
were analyzed and there was a statistically significant difference between group E2 and group
E1 (p < 0.05), and between group E2 and group NE (p < 0.005), whereas no significant
difference was found between groups E1 and NE. In group E2, total dyschromatopsia correlated
significantly with toluene in ambient air and in blood (both p < 0.05) as well as with hippuric acid
in urine after the work shift (p < 0.001). This study indicates that toluene can impair color vision
in exposed workers, and provides a NOAEL of 35 ppm (132 mg/m3) and a LOAEL of 156 ppm
(590 mg/m3). The authors did not comment on any potential gender-related differences in
acquired color vision impairment, even though Group E1 consisted of mostly female workers
and Group E2 consisted of mostly male workers.
Zavalic et al. (1998b) examined the effects of chronic occupational toluene exposure on color
vision using a group of 45 exposed male workers (mean toluene exposure concentration =
119.6 ppm (450 mg/m3), duration = 16.8 ± 5.94 yr) and 53 controls. Although not specified in the
study, the workers appear to be a sub-group of the same workers investigated in the other
Zavalic et al. (1998a, c) studies. Color vision was evaluated using the Lanthony desaturated
panel D-15 test on Wednesday morning before work and repeated on Monday morning, at least
64 hours after exposure; test scores were age and alcohol consumption-adjusted. Color vision
was significantly impaired in toluene-exposed workers (p < 0.0001) compared to controls. There
was no significant difference between test scores of the exposed workers on Monday morning
(prework) and Wednesday morning. The authors stated that the effect of toluene on color vision
can be chronic with a recovery period of possibly longer than 64 hours.
Cavalleri et al. (2000) evaluated color vision impairment in 33 toluene-exposed workers.

Toluene exposure was estimated to be 42 ppm by measuring urinary excretion of the
unmetabolized toluene (i.e. TolU). Color vision was tested with the Lanthony D-15 desaturated
panel, and the outcomes were expressed quantitatively with two indices of color perception, the
color confusion index (CCI) and the total confusion index (TOTCI). Toluene-exposed workers
had a subclinical reduction in color vision, compared with 16 referents (p < 0.01 and p < 0.001
for CCI and TOTCI, respectively). This effect was found to be related to cumulative solvent
exposure - estimated as the product of urinary excretion of unmodified toluene and previous
toluene exposure duration (Cavalleri et al., 2000). This analysis supported the hypothesis that
color vision impairment progresses as exposure continues. In the examined group of workers,
toluene exposure was within the occupational limit (50 ppm, 190 mg/m3) proposed by the
American Conference of Governmental Industrial Hygienists (ACGIH, 1997). The observed loss
in color vision raised doubts on the protection afforded by this limit with respect to the color
vision impairment health endpoint.
Campagna et al. (2001) examined 72 printers exposed to an average of 36 ppm (136 mg/m3)
toluene, 34 workers exposed to an average ambient concentration of 9 ppm (32 mg/m3) toluene,
and 19 non-exposed workers (controls) for acquired color vision loss analysis. The three groups’
average employment durations were 18, 19 and 8 years, respectively. Age and alcohol
consumption, both color vision-dependent factors, were controlled for in the study. Color vision
was assessed with the Lanthony D-15 desaturated panel method. Workers from the two
exposed groups had significantly higher CCI values than from the control group (p < 0.01,
Wilcoxon rank test). The authors suggested that the Lanthony D-15 desaturated panel detects
early neurotoxic effects among workers exposed to toluene.
Schaper et al. (2004) conducted a four-year study of three repeated examinations on correlation
between human occupational exposures to toluene and color vision impairment. A total of 189
workers were grouped as “high-level” (printing, mean current exposures of 26 ± 21 ppm (98 ±
79 mg/m3)) or “low-level” (end-processing, mean current exposures of 3 ± 4 ppm (11 ± 15
mg/m3)), with mean exposure durations of 23 ± 6 years (long-term) or 7 ± 2 years (short-term).
Color vision was tested with Lanthony desaturated color vision test D-15d and a CCI was
calculated. Repeated analyses of covariance (complete repeated dataset of 162 subjects) and
multiple regressions (highest available subjects of 267) did not demonstrate a significant effect
of toluene on color vision function.
Paramei et al. (2004) performed a meta-analysis of chronic toluene exposure on human color
vision impairment using effect sizes approach, which requires means and standard deviations
from the individual studies. Among the 11 studies from existing peer-reviewed human studies, 4
studies were included in the meta-analysis as fulfilling all three criteria: (1) use of the common
color discrimination test Lanthony Panel D-15d; (2) arithmetic means and standard deviations of
CCI available for both an exposed and unexposed groups; (3) documentation of exposure level
for the exposed group. The meta-analysis results showed generally higher CCI values for the
exposed groups and positive effect sizes for 3 of the 4 studies for toluene, indicating that color
discrimination was inferior for exposed groups in the majority of the studies. By applying a
random effects model, an average effect size of 0.15 was obtained (p = 0.44) with a weighted
mean exposure of 30 ppm of toluene, indicating an inferior performance of the exposed
subjects. None of the values reached significance at the 5%-level.
Auditory effects
Abbate et al. (1993) evaluated alterations induced in the auditory nervous system by exposure
to toluene in a group of rotogravure workers. A sample of 40 workers of normal hearing ability
was selected from a group of 300 workers who were apparently in good health but were
professionally exposed to toluene (12 – 14 years exposure, 97 ppm (370 mg/m3) average
exposure). They were subjected to an adaptation test utilizing a BAEP (Brainstem Auditory
Evoked Potential) technique with 11 and 90 stimulus repetitions a second. The results were
compared with an age and sex-matched control group not professionally exposed to solvents. A
statistically significant alteration in the brainstem auditory evoked response (BAER) results was
noted in the toluene-exposed workers with both 11 and 90 stimuli repetitions. Since alterations
of the BAEPs have been demonstrated to be positively correlated to otoneurotoxicity in animals,
the authors suggested that these results can be explained as a toluene-induced effect on the
auditory system, even in the absence of any clinical sign of neuropathy. Furthermore, this effect
was observed in the responses of the entire auditory system, from peripheral receptors to
brainstem nuclei.
Morata et al. (1997) studied the effects of organic solvents (mainly toluene) and noise on the
hearing of rotogravure printing workers. Pure-tone audiometry and immittance audiometry
testing were conducted with 124 workers who were occupationally exposed to various levels of
noise and an organic solvent mixture of toluene, ethyl acetate, and ethanol. The subjects were
fairly young employees, with the following average characteristics: age of 33.8 ± 8.5 years,
working tenure of 7.7 ± 6.1 years, noise exposure of 7.7 ± 6.0 years, and solvent exposure of
6.5 ± 6.0 years. Using biological monitoring of hippuric acid and creatinine in the urine, 109
solvent-exposed workers had their total toluene exposure assessed for statistical analysis. The
levels of toluene in the air ranged from 0.14 to 919 mg/m3 (0.037 to 244 ppm). The measured
toluene air concentrations and levels of toluene urinary metabolites were found to be correlated.
The results of this study showed that forty-nine percent of the workers had hearing loss. Among
the numerous variables that were analyzed, age, and toluene urinary metabolites were shown to
be statistically significant on the development of hearing loss. The findings suggested that
exposure to toluene has a toxic effect on the auditory system. However, the authors did not
identify the specific concentration of urinary hippuric acid corresponding to a NOAEL or LOAEL
for the end point of hearing loss, and no control group was used for comparison.
Schaper et al. (2003, 2008) studied the ototoxicity of occupational exposure to toluene below 50
ppm (188 mg/m3) with 333 male workers from rotogravure printing plants. The mean past
lifetime weighted average exposures (LWAE) measures to toluene and noise were 45 ± 17 ppm
(170 ± 64 mg/m3) and 82 ± 7 dB(A) for high-toluene-exposed printers, and 10 ± 7 ppm (38 ± 26
mg/m3) and 82 ± 4 dB(A) for low-toluene-exposed end-processors. The mean current exposures
to toluene and noise were 26 ± 20 ppm (98 ± 75 mg/m3) and 81 ± 4 dB(A) for printers, and 3 ± 3
ppm (11 ± 11 mg/m3) and 82 ± 4 dB(A) for end-processors. The auditory thresholds were
measured with pure tone audiometry. Statistical analyses did not reveal significant effects of
toluene concentration, exposure duration and interactions between toluene intensity and noise
intensity. In this study, 45 ppm (169 mg/m3) was a NOAEL.
Neuroendocrine effects
Svensson et al. (1992a) studied the neuroendocrine effect of toluene on the plasma
concentration of LH and testosterone in 47 male workers from two rotogravure printing
companies, with 46 metal workers as a control group. The average toluene exposure at the two
printing companies was 11 ppm (41 mg/m3) (1–108 ppm (4–410 mg/m3)) and 47 ppm (180
mg/m3) (6–142 ppm (23–550 mg/m3)) at the time of study sampling, respectively. The time
weighted average air toluene concentration was below 80 ppm (300 mg/m3) (Swedish threshold
limit value) for all 47 subjects, while the median historical cumulative exposure (ppm x years)
was 2,896 ppm-yr. Increasing exposures were significantly associated with decreasing plasma
concentration of LH (p = 0.003) and testosterone (p = 0.02). Cumulative exposure had no
correlation with plasma hormone concentration. The authors concluded that low toluene
exposure had an effect on the hypothalamus-pituitary axis.
Svensson et al. (1992b) also studied the neuroendocrine effects of occupational toluene
exposure in a different group of rotogravure male printing workers (n = 20, exposure group),
compared with 44 male industrial workers without toluene exposure (control group). The median
individual time-weighted toluene concentration in air was 36 ppm (136 mg/m3) (range 8–111
ppm (30–420 mg/m3)), while the median historical cumulative exposure was 5,630 ppm-year for
the printing workers. The hormone assays showed lower median plasma levels of FSH (p =
0.02) and LH (p = 0.05), and also lower serum level of free testosterone (p = 0.05) for the
exposure group, but the decreases of hormone levels were not significantly correlated to
airborne toluene concentration. In 8 out of the 20 exposed workers, the levels of FSH and LH
increased during a 4 week vacation, which the authors concluded was a slight and reversible
effect of toluene on pituitary function in addition to a general depression of brain functions.
However, no solvent-induced cases of toxic encephalopathy in the exposed group were verified.
Other chronic effects

Wang et al. (1996) showed decreased liver and kidney function in workers due to exposure to
low concentrations of toluene. The liver function test results for 153 workers (108 males and 45
females) exposed to 1.0-60.4 ppm (4.0–228 mg/m3) toluene for at least 2-5 years (male and
female test groups) were compared with those for 420 workers (238 males and 182 females)
who had never been occupationally exposed to solvents (male and female control groups). The
results showed significantly lowered serum glutamic-pyruvic transaminase (GPT) and gamma-
glutamyl transpeptidase (γ-GTP) activities in the male test group, but significantly higher serum
GPT and γ-GTP activities in the female test group, compared to those in their respective control
groups. These effects suggested that toluene exposure below 100 ppm (380 mg/m3) was
possibly causing the changes in liver function. Body mass index and alcohol consumption were
ruled out as possible confounders for the observed difference. However, the authors suggested
that other life-style factors and nutritional status may be factors for liver enzyme differences
between groups.
The studies investigating long-term exposure of toluene in humans are summarized below in
Table 3.
Table 3. Summary of toluene occupational exposure chronic studies in adult workers

with identified NOAEL and/or LOAEL.
Exposure
Duration NOAEL LOAEL
Number of (average Concentration Critical Effects ppm ppm
Studies Subjects years ± SD) (TWA in ppm) (endpoints) (mg/m3) (mg/m3)
Yin (1987) 101 exposed, 6.8 42.8 Sore throat, dizziness, * 42.8
127 controls headache (161)
Orbaek & 30 pooled 29 (median) 11.4 (A) Fatigue, memory * <41.8
Nise workers from 41.8 (B) problems, concentration (158)
(1989) two shops, A difficulties.
(n = 19) and B Marginal effects on
(n = 11). performance in
50 controls psychometric tests
Foo 30 exposed, 5.7 ± 3.2, 88 (exposed), Significant decrease in * 88
(1990) 30 controls 2.5 ± 2.7 13 (controls) neurobehavioral (330)
performance (6 out of 8
tests)
nd *
Nakatsuka 174 exposed, 46 (geometric No measured effect on 46
(1992) 120 controls mean), color vision (170)
0 ppm
Abbate 40 exposed, 12-14 97, Auditory nervous system * 97

(1993) 40 controls 0 ppm (BAER) 28% increase of (370)
latency shift
Vrca et al. 59 unexposed 0 ppm for 0 ppm, or 80 In exposed workers, ND 60-80
(1995) workers (54 20.6 ± 7.7 ppm higher VEP wave (230-
males, 5 years, or 60- amplitudes and longer 300)
females), age 80 ppm for wave latency indicative of
43 ± 7.2 years 21.4 ± 7.4 more excitable wave
or 49 toluene- years generators in the visual
exposed pathway and reduced
workers (46 wave conductivity in
males, 3 certain segments of the
females), age visual pathway,
42.3 ± 6.8 respectively.
years
Vrca et al. Same as Same as Same as Longer BAEP wave Same Same
(1996) above above above latencies and smaller as as
wave amplitudes in above above
exposed versus
unexposed workers
suggesting changes to the
auditory pathway.
Vrca et al. Same as Same as Same as Increased VEP wave Same Same
(1997b) above above above latency during mental as as
engagement suggesting above above
adverse changes to
intracranial structures like
the brainstem and
hypothalamus.
Abbreviations: BAEP – brainstem auditory evoked potential; BAER – brainstem auditory evoked response; ND – not
determined; VEP – visual evoked potential.
* not observed
nd No data
Table 3. Summary of toluene occupational exposure chronic studies in adult workers

with identified NOAEL and/or LOAEL (continued).
Exposure
Duration NOAEL LOAEL
Number of (average Concentration Critical Effects ppm ppm
Studies Subjects years ± SD) (TWA in ppm) (endpoints) (mg/m3) (mg/m3)
Vrca 49 exposed, 21.6 40-60 (est.), ** BAEP evoked auditory * 40-60
(1997a) 59 controls 0 ppm potential increase in (150-
latencies, dose-response 230)
Boey 29 exposed, 4.9 ± 3.5 90.9, Significant decrease in * 90.9
(1997) 29 controls 0 ppm neurobehavioral (340)
performance (9 of 14
tests)
Zavalic 83 controls, 20.91 (work), 0 ppm, Color vision impairment in 35 156
(1998a,c) 1 low exp, 15.60 ± 4.61, 35 (11.3-49.3), high exposure group (130) (590)
32 high exp 19.86 ± 5.61 156 (66-250)
Zavalic 53 controls, 22.4 (work), 0 ppm, Color vision impairment in * 120
(1998b) 45 male 16.8 ± 5.94 120 exposed group (450)
workers
exposed
Eller 19 controls, 7.7 ± 3.5 0 ppm, No difference between <20 >100
(1999) (range:1-12), low exposure group and (75) (380)
30 with low 25.5 ± 8.9 <20, control group; impaired
exposure, (range:13-40) neuropsychological
49 with high >100 function in high exposure
exposure group
Cavalleri 33 exposed, 9.75 42 (est.) ** Increase in color vision * 42
(2000) 16 controls impairment test index (160)
Chouani 128 workers 14 ± 10 0-27 Subjective symptoms and 27 *
ere et al. (114 male, 14 psychometric testing (102)
(2002) female)
Zupanic 154 high-dose 15.3 ± 9.7 High 45.1 ± psychomotor performance 45 *
et al. 124 low-dose 14.5 ± 8.6 16.4, and subjective symptoms (169)
(2002) Low 9.3 ± 7.6
Schaper 47 short-low, 6.3 ± 2.3, Hi 44.7 ± 17.1, ototoxicity 45 *
et al. 39 long-low, 21.4 ± 7.4, Low 9.5 ± 7.3 (169)
(2003, 59 short-high, 5.6 ± 3.8,
2008) 47 long-high 22.0 ± 7.5
Schaper 41 short-low, 8.11 ± 2.23, Hi 42.66 ± Color vision impairment 43 *
et al. 28 long-low, 21.82 ± 5.31, 15.71, (162)
(2004) 54 short-high, 6.82 ± 2.42, Low 9.16 ±
39 long-high 22.36 ± 7.44 7.90
Seeber 47 short-low, 6.6 ± 2.1, Hi 45 ± 17, Psychological tests and 45 *
et al. 39 long-low, 21.3 ± 6.0, Low 9 ± 7 psychomotor functions (169)
(2004, 59 short-high, 5.5 ± 3.6,
2005) 47 long-high 21.2 ± 7.0
Kang et 20 high, 5.4 ± 5.3, High 70-80, Neurobehavioral tests ~20 ~75
al. (2005) 13 medium, 8.3 ± 9.5, Medium 20-30, (75) (282)
21 low 10.9 ± 6.4 Low < 1
Abbreviations: BAEP - brainstem auditory evoked potential; ND – not determined; VEP – visual evoked potential.
* not observed; ** indirect exposure estimate: based on urinary levels of metabolites and toluene blood levels for the Vrca 1997a
study, and based on urinary excretion of toluene plus data from previous air toluene measurements for the Cavalleri study.
Chronic Toxicity to Infants and Children

Several studies examined the health impact of ambient exposures of children to mixtures of air
toxics including toluene (below the U.S. EPA RfC of 5.0 mg/m3 (1.3 ppm)). Individual study
summaries can be found under “Community Epidemiological Studies” in section 6.1 above.
Since these studies are examinations on ambient mixtures of air toxics, they are less likely to
provide data for developing RELs.
Toluene abuse by pregnant women has resulted in infants born with phenotypic features similar
to fetal alcohol syndrome, such as postnatal growth deficiency, craniofacial malformations and
hydronephrosis (Hoyme et al., 1993) and is discussed in Section 7.1 below.
Chronic Toxicity to Experimental Animals
Neurobehavioral effects
Toluene affects dopamine levels in the brain, the key neurotransmitter of working memory in the
prefrontal cortex. Toluene has been implicated for many years in the changes of dopamine
levels in the brain, as measured in exposed neonatal and adult animals (von Euler et al., 1989;
Riegel et al., 2004) or by toluene’s effects on dopamine-dominated functions, such as hyper-
locomotion (von Euler et al., 1993; Riegel and French, 1999; Riegel et al., 2003) and activation
of mesolimbic reward pathways (Riegel and French, 2002).
Effects on the CNS have been observed in studies of animals chronically exposed to toluene by
inhalation. Inflammation and degeneration of the nasal and airway epithelium and pulmonary
lesions have also been observed in rats and mice chronically exposed to high levels of toluene
by inhalation. Adverse effects on the liver, kidneys, lungs and auditory system (i.e., high-
frequency hearing loss) have been reported in some chronic inhalation studies of rodents.
In a comprehensive chronic exposure study, male and female Fischer-344 rats (120/group/sex)
were exposed to 30, 100, or 300 ppm (110, 380, or 1100 mg/m3) toluene for 6 hrs/day, 5
days/week for two years (Gibson and Hardisty, 1983). Pathologic, hematologic, clinical blood
chemistry, urinalysis, and ophthalmologic examination did not reveal any injury considered to be
evidence of chemical toxicity. Body weights were elevated in exposed male and female rats, but
no clear-cut dose response relationship was apparent.
A persistent increase in the affinity of dopamine D2 agonist binding in the rat caudate-putamen
was observed with exposure to 80 ppm (300 mg/m3) 6 hr/day, 5 days a week for 4 weeks (TWA
14 ppm/week), followed by a post-exposure period of 29–40 days. However, similar exposure of
adult rats to 40 ppm (150 mg/m3) toluene for 4 weeks did not result in these specific effects
(Hillefors-Berglund et al., 1995). In contrast, longer duration (16-week, 104 hr/week) inhalation
exposure of adult rats to 40 ppm (150 mg/m3) toluene produced behavioral neurotoxicity and
alterations in neurotransmitters (Berenguer et al., 2003). Neurobehavioral alterations were
determined by assaying locomotor activity and rearing activity. Both exposed males and
females had significant differences in rearing activity compared to control animals.
Dopaminergic and serotoninergic neurotransmission activity was significantly altered in various

brain regions of rats exposed to 40 ppm (150 mg/m3) toluene for 16 weeks.
Macaque monkeys were exposed by inhalation for 50 minutes to one of six concentrations of
toluene (0, 100, 200, 500, 1000, 2000 or 4500 ppm; 6 animals/treatment group) twice/week for 6
weeks (Taylor and Evans, 1985). Attention deficits and impairment of cognitive and motor
abilities were observed beginning at 2,000 ppm (7,500 mg/m³) using a repeated measures
analysis of variance test with a statistical significance of p < 0.05. Expired carbon dioxide
showed an inverted-U shaped response initially increasing above control levels, and then
decreasing below control levels (primarily at 4,500 ppm (17,000 mg/m³)) with increasing toluene
concentration. The authors noted this type of curve suggests behavioral stimulation at lower
concentrations and behavioral sedation at higher concentrations.
Toluene exposure produced neurological impairments in the ability to create new strategies
after investigators made changes in the presence or placement of the hidden platform in the
Morris water maze (Hass et al., 1999; Hougaard et al., 1999; von Euler et al., 2000). These
studies were performed on young rats with prenatal, postnatal, and adolescent exposure, and at
widely varying doses. The lowest dose tested in the Morris water maze assay was 80 ppm (300
mg/m3) for 4 weeks in adolescent rats in the study by von Euler et al. (2000), which also caused
significantly reduced performance in beam-walk test, used to detect neurological deficits in
sensory, balance, or motor performance (detailed below).
Hass et al. (1999) exposed female rats to 0 or 1200 ppm (4500 mg/m3) toluene for 6 hours per
day from day 7 of pregnancy until day 18 postnatal. Developmental and neurobehavioral effects
in the offspring were investigated using a test battery including assessment of functions similar
to those in the proposed Organization for Economic Cooperation and Development (OECD)
Testing Guidelines for Developmental Neurotoxicity Study (OECD, 2006) (physical
development, reflex development, motor function, motor activity, sensory function, and learning
and memory). The exposure did not cause maternal toxicity or decreased offspring viability.
However, lower birth weight, delayed development of reflexes, and increased motor activity in
the open field were noted in the exposed offspring. The exposed female offspring had poorer
scores on a Morris water maze test (they took longer to locate a hidden platform after platform
relocation) at the age of 3.5 months indicating impaired cognitive function. The difference was
not related to impaired swimming capabilities since swim speeds were similar to control values.
The authors stated that exposure to 1200 ppm (4500 mg/m3) toluene during brain development
caused long-lasting developmental neurotoxicity in rats.
Hougaard et al. (1999) studied the development and neurobehavioral effects of prenatal
exposure to toluene by exposing 16 pregnant rats (Mol:WIST) to 1800 ppm (6800 mg/m3) of
toluene in whole body inhalation chambers for 6 h per day, 2 weeks on days 7–20 of gestation.
Body weights of exposed pups were lower until day 10 after parturition. Neurobehavioral tests
included neuromotor abilities (rotarod), activity level (open field), reactivity, habituation and
prepulse inhibition (acoustic startle), sensory function (auditory brain stem response), and
learning and memory ability (Morris water maze). Evaluation of the pups revealed no effects on
motor function, activity level, acoustic startle, and prepulse inhibition. Auditory brain stem
response measurements of hearing function revealed small effects in male exposed offspring.
Morris water maze performance during initial learning indicated some impaired cognitive
functions and was confirmed during further testing, especially in reversal and new learning.
Effects on cognitive functions seemed most marked in female offspring.
Von Euler et al. (2000) investigated the effect of inhalation exposure to 0 ppm and 80 ppm (0
and 300 mg/m3) toluene for 4 weeks (6 hr/day, 5 days/week) on the behavior and brain features
in 60 male Sprague-Dawley rats (about 50 days of age). At least 4 weeks after the final
exposure, the rats’ spatial memory was altered by toluene, in that they spent a longer time in the
initial quadrant of a Morris swim maze. Toluene-exposed rats also showed trends for increases
in both locomotion and rearing behaviors and a significantly reduced beam-walk performance.
Magnetic resonance imaging of living rats and autoradiograms of frozen brain sections showed
a decreased area of the cerebral cortex, especially the parietal cortex, by 6–10%. The
biochemical receptor binding assays indicated a persistent effect of toluene selectively binding
to dopamine D2 receptors. They concluded that low concentrations of toluene exposure led to
persistent effects on cognitive, neurological, and brain-structural properties in the rat.
Bowen and McDonald (2009) studied the behavioral effects of repeated toluene binge exposure
(high dose of toluene as to mimic toluene abuse) on cognitive function (behavioral impulse
control) of Swiss Webster mice using a “wait-for-reward” operant task. After being trained on
fixed-ratio schedule wait task, groups of 40 mice were exposed to 1000, 3600 or 6000 ppm
(3800, 13,600, or 22,600 mg/m3) toluene for 30 min per day for 40 days. Repeated toluene
exposure decreased response rates and resulted in a higher response-to-reinforcer ratio than
the control group exposed to air for the same duration. Mice receiving the highest exposure
level (6000 ppm (22,600 mg/m3)) showed a dramatic decrease in the number of rewards
received, while those exposed to 3600 ppm (13,600 mg/m3) of toluene had significantly more
responses. Mice exposed to the lowest level (1000 ppm (3800 mg/m3)) showed little change in
the number of rewards. The authors concluded that repeated binge exposures to high
concentrations of toluene can significantly interfere with behavioral performance, suggesting a
significant impact on cognitive and/or psychomotor function.
To determine the neurobehavioral effects of subchronic exposure to toluene, groups of 160

adult male Long-Evans rats inhaled toluene vapor (0, 10, 100, or 1000 ppm (0, 38, 380, or 3800
mg/m3)) for 6 hr/day, 5 days/week for 13 weeks and were evaluated on a series of behavioral
tests beginning 3 days after the end of exposure (Beasley et al. 2010). Toluene delayed
appetitively-motivated acquisition of a lever-press response in all treatment groups (p < 0.05),
but did not affect the responses on motor activity, anxiety-related behavior in the elevated plus
maze, trace fear conditioning, acquisition of an appetitively-motivated visual discrimination, or
performance of a visual signal detection task. They concluded that these results were consistent
with a pattern of subtle and inconsistent long-term effects of daily exposure to toluene vapor, in
contrast to robust and reliable effects of acute inhalation of toluene.
Other CNS effects

Airborne pollutants and toxics such as toluene interact with chemoreceptors in the nasal cavity,
especially trigeminal and olfactory receptors. To elucidate the influence of toluene inhalation on
the mitral and granular neurons in olfactory bulbs and the pyramidal cells in hippocampus of
rats, Gelazonia et al. (2006a, 2006b) exposed two age groups of rats (one and two months old),
5 per group, to air saturated with toluene vapor (concentration unknown) in closed glass
chambers for 40 days (6 days/week, 3-4 minutes per day until the rats attained a sidewise
laying position). Another two groups of rats, 5 each, of either one or two months of age, were
exposed to air only, as control groups. The results showed that, compared with the respective
control groups, the exposed rats had a significantly decreased number of mitral neuron cells
(43% reduction for one-month rats and 28% reduction for two-month ones), while the granular
cells remained unaltered in both age groups of exposed rats. The number of pyramidal neurons
in the hippocampus decreased by 26% in one-month exposed rats only, which induced
deterioration of the hippocampal neural circuits and destruction of memory and learning
processes.
To evaluate the potential modifications of subchronic exposure to inhaled toluene on behavior

and olfactory functioning, Jacquot et al. (2006) exposed mice to 1000 ppm (3800 mg/m3) of
toluene for 5 hr/day, 5 days/week for 4 weeks, and assessed their behavioral (sensitive and
perceptive) and histological (cellular level) changes. Tests were administered during the 4-week
exposure (W1-W4) and up to 4 weeks following exposure (W5-W8). Behavioral evaluation (T-
maze test) of mice sensitivity toward toluene (as a repulsive odor) showed a constant decrease
(less sensitive) during the 4 weeks of exposure, which continued for 2 weeks after the exposure
(W5, W6). During the last two weeks of the study (W7, W8), the sensitivity of mice to toluene
returned to normal. On the cellular level, the density of olfactory epithelium cells decreased
markedly during W3 and W4 and increased significantly in the first week of the recovery period
(W5). The thickness of olfactory neuroepithelium decreased at W1, followed by an increase at
W2 and W3 (suggesting an inflammatory process), but decreased abruptly at W4, followed by a
gradual return to normal at W5 through W8.
Respiratory effects
The National Toxicology Program exposed F344/N rats and B6C3F1 mice (60 males and 60
females of each species) to toluene 6.5 hrs/day, 5 days/week for up to two years (NTP, 1990)
Toluene levels were 600 and 1200 ppm (2300 and 4500 mg/m3) for rats, and 120, 600 and 1200
ppm (450, 2300 and 4500 mg/m3) for mice. At the 15-month interim sacrifice, incidences and
severity of nasal cavity lesions, including degeneration of olfactory and respiratory epithelium
and goblet cell hyperplasia, were increased in exposed rats at both dose levels. Minimal
hyperplasia of the bronchial epithelium was seen in female mice at 1200 ppm (4500 mg/m3).
Severity of nephropathy, but not incidence, was slightly increased in exposed female rats at
both dose levels. Following two years of exposure, erosion of olfactory epithelium and
degeneration of respiratory epithelium were increased in exposed rats. Inflammation of nasal
mucosa and metaplasia of olfactory epithelium were increased in exposed female rats. These
lesions were not seen in mice. No biologically important non-neoplastic lesions were observed
in mice. Nephropathy was seen in almost all rats, and severity was somewhat increased in
exposed rats.
To investigate the effect of long-term, low level toluene exposure on airway inflammatory
responses in mouse lung, Fujimaki et al. (2007) exposed female C3H mice to 0 or 50 ppm (0 or
190 mg/m3) of toluene or air for 6 hr/day on 5 days/week for 6 or 12 weeks in whole-body
exposure chambers. One day after the last toluene exposure, they collected bronchoalveolar
lavage (BAL) fluid from each mouse and examined cellular infiltration and production of
cytokines, chemokines, neurotrophins and substance P with the ELISA method. They found that
the number of total cells and macrophages increased significantly (p < 0.05) in mice of both 6-
and 12-week-exposure. The production of interferon-gamma and substance P were decreased
significantly. In addition, neurotrophin-3 production in BAL fluid was significantly increased only
in 12-week-exposed mice. This study suggested long-term, low-level toluene exposure
modulates airway inflammatory response in mice through neurological signaling.
Shwe et al. (2007) exposed male C3H mice to 0, 9 and 90 ppm (0, 34 and 340 mg/m3) toluene
for 30 min by nose-only inhalation on days 0, 1, 2, 7, 14, 21, and 28 to study the effects of
volatile organic compounds in the indoor air on the induction or augmentation of airway
inflammatory responses (general neuroimmune interaction). One day after the 28-day exposure
period, bronchoalveolar lavage (BAL) fluid was collected for analysis of inflammatory cell influx,
while lung tissue and blood samples were collected to determine cytokine, neurotrophin mRNA,
protein expressions, and plasma antibody titers. Exposure of mice to 9 ppm (34 mg/m3) or 90
ppm (340 mg/m3) toluene both resulted in increased inflammatory cell infiltration in BAL fluid,
increased IL-5 mRNA, decreased nerve growth factor receptor tropomyosin-related kinase A
and brain-derived neurotrophic factor mRNAs in lung, and increased IgE and IgG1 antibodies
and nerve growth factor content in the plasma. Even though there was no pathological endpoint
in this study, these findings suggested that low-level toluene exposure aggravates the airway
inflammatory responses.
Auditory Effects
Hearing loss was observed in groups of rats after exposure to various toluene exposure
scenarios: 1000 ppm (3800 mg/m³), 14 hours per day for 2 weeks; 1500 ppm (5700 mg/m3) for
14 hours per day for three days; 2000 ppm (7500 mg/m3) for 8 hours per day for three days; and
intermittent exposure to 3000 ppm (11,300 mg/m3) for 30 minutes every hour, 8 hours per day
for 2 weeks (Pryor et al., 1984). However, groups of rats exposed to lower concentrations (400
and 700 ppm (1500 and 2600 mg/m3)) for 14 hours per day did not have hearing loss even after
16 weeks of exposure, and single exposures to 4000 ppm (15,100 mg/m3) for 4 hours or 2000
ppm (7500 mg/m3) for 8 hours were also not ototoxic.
Toluene has been shown to disrupt the auditory system in rats but not in guinea pigs, whose
high amount of hepatic cytochrome P-450s and high concentration of glutathione in the cochlea
likely play a key role in its auditory resistance to long-term, high-level toluene exposure.
Waniusiow et al. (2009) tested toluene-induced hearing loss in glutathione-depleted guinea pigs
whose P-450 activity was partly inhibited. The animals were exposed to 1750 ppm (6600
mg/m3) toluene 6 hr/day, 5 days/week for 4 weeks. Auditory function was tested by
electrocochleography and supported by subsequent histological examination. The authors
noted that a significant toluene-induced hearing loss was provoked in these exposed guinea
pigs, but the associated auditory system histopathology has not been demonstrated in studies
using other toluene-exposed species, including rats. Histological examination showed that only
the stria vascularis and the spiral fibers were disrupted in the apical coil of the cochlea of the
guinea pigs. The authors concluded that guinea pigs can metabolize toluene more efficiently
than rats, probably because of a higher level of hepatic P-450.
Table 4. Summary of chronic animal studies with identified NOAEL and/or LOAEL.
Study Exposure duration and Subjects / Effects (endpoints) NOAEL LOAEL

Concentration (ppm) ppm ppm
(mg/m3) (mg/m3)
Gibson and 30, 100, or 300 ppm 6 male and female Fischer-344 rats 300 NA
Hardisty hrs/day, 5 days/week for (120/group/sex) / no effect on (1130)
(1983) two years pathology, blood chemistry, urinalysis,
and ophthalmologic examination
Taylor and 0, 100, 200, 500, 1000, Macaque monkeys / Attention deficits 1000 2000
Evans 2000 or 4500 ppm for 50 and impairment of cognitive and motor (3800) (7600)
(1985) min/day, 2x/week for 6 abilities
weeks
Von Euler et 0 ppm and 80 ppm for 4 60 male Sprague-Dawley rats / Altered NA 80 (300)
al. (2000) weeks (6 hr/day, 5 spatial memory and changes in brain
days/week) structural features
Beasley et 0, 10, 100, or 1000 ppm 160 adult male Long-Evans rats / NA 10 (38)
al. (2010) for 6 hr/day, 5 days/week battery of neurobehavioral tests – only
for 13 weeks finding was delayed appetitively-
motivated acquisition of a lever-press
response
Jacquot et 1000 ppm for 5 hr/day, 5 Mice / behavioral (sensitive and NA 1000
al. (2006) days/week for 4 weeks perceptive) and histological (cellular (3800)
level) changes
NTP (1990) 600 and 1200 ppm for F344/N rats and B6C3F1 mice (60 NA for 600 for
rats, and 120, 600 and males and 60 females of each species) rats, 600 rats
1200 ppm for mice, 6.5 / nasal cavity lesions, erosion of for mice (2300),
hrs/day, 5 days/week for olfactory epithelium and degeneration (2300) 1200 for
up to two years of respiratory epithelium female
mice
(4500)
Fujimaki et 0 or 50 ppm for 6 hr/day C3H mice / cellular infiltration in NA 50 (190)

al. (2007) on 5 days/week for 6 or bronchoalveolar lavage (BAL) fluid
12 weeks
Shwe et al. 0, 9 and 90 ppm for 30 C3H mice / induction or augmentation NA 9 (34)
(2007) min by nose-only of airway inflammatory responses in
inhalation on days 0, 1, 2, BAL
7, 14, 21, and 28
Waniusiow 1750 ppm for 6 hr/day, 5 glutathione-depleted guinea pigs / NA 1750

et al. (2009) days/week for 4 weeks Auditory function loss (6580)
Other chronic effects

To investigate the adverse effects of toluene inhalation on bone morbidity and bone
mineralization, Atay et al. (2005) exposed 10 4-wk-old male Swiss albino BALB/c mice to 300
ppm (1100 mg/m3) (static) toluene 6 hr per day for 8 weeks and measured the bone mineral
density and bone mineral content in the femoral neck by dual X-ray absorptiometry bone
densitometer. They found that the bone mineral density was significantly reduced compared to a
control group of another 10 mice of the same type. They concluded that chronic exposure to
toluene affected bone metabolism and could contribute to bone resorption and inhibition of bone
formation.
Toluene has also been shown to impair visual functions in animal studies. Boyes et al. (2016)
examined visual function of male Long-Evans rats by recording visual evoked potentials (VEP)
and / or electroretinograms (ERG) in four sets of experiments. First set exposed 40 rats per
group to 0, 10, 100, 1000 ppm (0, 38, 380, 3,800 mg/m3) toluene in controlled inhalation
chambers, 6h/d 5d/wk for 13 weeks, and one week after the exposure completion their VEPs
were recorded, which were not significantly changed by toluene exposure. Four to five weeks
after exposure ended, their ERGs were recorded and showed that only the visual function of
rats exposed to 1000 ppm (3800 mg/m3) toluene were reduced.
A second set of approximately 40 rats per group were exposed concurrently with the first set for
13 weeks. One year after the exposure ended, their ERGs were recorded and again only rats
exposed to 1000 ppm (3800 mg/m3) toluene were shown to have visual function negatively
affected. A third set of approximately 40 rats per group were exposed to the same
concentrations of toluene for 4 weeks.
A fourth set of approximately 20 rats per group exposed to 0 or 1000 ppm (0 or 3800 mg/m3)
toluene for 4 weeks were tested 1 year after the exposure termination. ERGs of rats exposed for
4 weeks were not significantly reduced. The reductions of ERGs after 13 weeks of exposure
and persisting for 1 year suggest alterations in rat retina. The authors concluded that repeated
toluene exposure may lead to subtle persistent changes in rat visual function, and particularly
the rat retina may be more sensitive to toluene exposure than visual cortex.
Young / Adult Animal Comparisons

Samuel-Herter et al. (2014) studied the possibility of age-dependent neurobehavioral sensitivity
to toluene among inhalant abusers using a rodent model to assess the effects of acute binge-
like toluene inhalation exposures. Male Long-Evans rats (18/exposure group) were exposed to
approximately 5000 ppm (18,800 mg/m3) for 15 or 30 min in whole-body exposure chambers.
The following age-groups of rats were used in the study: adolescent (1 month), young adult (2-3
months), adult (5-6 months), and older adult (10-12 months). Motor functions evaluated included
ambulatory activity, vertical exploration, grooming, balance, gait and neurological functions. An
adolescent group of rats were not exposed to 30 min of toluene due to a pilot study result
showing that rats in this age group required much longer time to recover any degree of motor
function (reference not given). The general results showed that acute toluene exposure
impaired both motor and neurological functions in all age groups of rats, with adolescent and
young adult rats needing significantly longer recovery times than older rats (p < 0.05). The
authors claimed that these results suggested an age-dependent vulnerability to the intoxicating
effects of toluene. However, the possible cause of the adolescent and young adult age-groups
of rats receiving higher doses (per unit body weight) was not discussed.
7. Developmental and Reproductive Toxicity

Human Studies
Toluene has been listed under Proposition 65 as known to the State of California to cause
developmental toxicity (OEHHA, 2015). Most of the information concerning the adverse
developmental effects of toluene in humans comes from case reports of children born to organic
solvent “sniffers”, in which toluene was often the primary solvent inhaled. Children whose
mothers had inhaled large quantities of toluene during pregnancy were found to have
microencephaly, facial and limb abnormalities, attention deficits, hyperactivity, developmental
delay with greater language impairment, and growth retardation similar to effects of alcohol
abuse (Hersh et al., 1985; Hersh, 1989).
In other studies, hyperchloremic acidosis along with growth retardation and craniofacial
abnormalities were observed in the children of women with severe renal tubular acidosis
induced by chronic paint sniffing (Goodwin, 1988). Preterm delivery, perinatal death, and growth
retardation were significantly increased in a study of 21 newborns exposed to toluene as a
result of maternal inhalation abuse (Wilkins-Haug and Gabow, 1991).
In a case referent study of women occupationally exposed to organic solvents including toluene,
increased incidences of urogenital, gastrointestinal, and cardiac anomalies were reported in
their children (McDonald et al., 1987). Paternal occupational toluene exposure (in which the
mothers had no exposure) increased the odds ratio for spontaneous abortions; however, these
observations cannot be clearly ascribed to toluene because of the small number of cases
evaluated and the large number of confounding variables (Lindbohm et al., 1992).
An increased incidence of spontaneous abortions was also reported among occupationally

exposed women. Ng et al. (1992) conducted a study on rates of menstrual disorders among
female workers exposed exclusively to toluene at a factory where audio coils and speakers
were assembled. The menstrual function questionnaire results of 231 female production
workers exposed to high toluene exposure (50-150 ppm (190-570 mg/m3), mean 88 ppm (330
mg/m3), average employment duration 6.0 years) were compared with those for 58 female
workers in the same factory with low toluene exposure (0-25 ppm (0-94 mg/m3), average
employment duration 6.7 years) (factory controls) and 187 working class women receiving
routine care at maternal and child health centers (external community controls). Dysmenorrhea
occurred more frequently in women exposed to high concentration of toluene compared with
external community controls (p < 0.001), but not compared to factory controls (not significant at
p < 0.05). The rates for dysfunctional uterine bleeding were similar in all groups, and there was
no evidence that dysfunctional uterine bleeding resulted from exposure to toluene.
An epidemiology study by Plenge-Bonig and Karmaus (1999) involved 150 male and 90 female
printers in Germany exposed to toluene, which were compared with non-exposed workers in
other industries using an infertility and subfecundity questionnaire. Historical exposure levels
varied from 10 to 200 ppm. The fecundability ratio (FR) was estimated on the basis of time to
pregnancy (TTP) or periods of unprotected intercourse not leading to pregnancy (PUNP). After
adjustment for confounders such as age, ethnicity, smoking, parity and pelvic inflammatory
diseases, 169 periods of TTP or PUNP in men and 100 periods in women were analyzed. The
results showed no increased risk to males at any exposure level (FR 1.05, 95% confidence
interval 0.93 to 1.19, p = 0.43), but fecundity was reduced in females by about half (FR 0.47,
95% confidence interval 0.29 to 0.77). The authors concluded that even though there were
possible biases (e.g., participant selection bias, recall bias), low daily exposure to toluene in
women seems to be associated with reduced fecundity, likely due to hormonal regulation
changes or early fetal loss.
The epidemiology study by Ghosh et al. (2012) examined the associations of low birth weight
(LBW) with toxic air pollutants in traffic exhaust including toluene. The data for 8181 children
with term LBW (>=37 weeks’ completed gestation and birth weight <2500 g) and 370,922 term
normal-weight children in Los Angeles (LA) Country were compared against land-use-based
regression (LUR)-modeled estimates and air toxics exposure, covering 1995 through 2006.
Measurements of air toxics including toluene were available for every 12 days from four
California Air Resources Board (CARB) air toxics monitoring stations in LA County and their
averages were calculated. The geocoded residential addresses of the mothers from the birth
certificates who resided ≤ 5 miles (8 km) from a CARB air toxics station were overlaid with the
LUR model to assign estimated exposures. The results showed BTEX exposures in the third
trimester and the last month of pregnancy were particularly associated with odds of term LBW,
while no association for first and second trimester and entire pregnancy exposure averages.
This study provided evidence for traffic exhaust including toluene’s contribution to term LBW.
Mothers who deliver at term have greater odds of delivering a low-weight baby when exposed to
higher levels of traffic exhaust pollutants including toluene in the third trimester.
Animal Studies
There are a number of older animal inhalation studies of varying quality investigating the
reproductive and developmental toxicity of toluene. The following animal studies support the
association between toluene exposure and effects on somatic development of the fetus.
However, the value of these studies is limited by issues such as unknown or unconventional
exposure durations, inadequate descriptions of maternal toxicity, use of individual offspring
instead of litters for statistical analyses, as well as questions about the presence of
contaminants in the toluene used (Donald et al., 1991).
Shigeta et al. (1982) reported that in the offspring of mice exposed by inhalation to 100 ppm
(380 mg/m³) and 1000 ppm (3800 mg/m³) toluene for 6 hours per day on days 1–17 of
gestation, the number of fetal resorptions increased. However, the increases showed neither a
dose-response nor were they statistically significant (no p-value given). Exposure at 1000 ppm
(3800 mg/m³) resulted in a statistically significant increase in the incidence of extra ribs. A
statistically insignificant increased incidence of extra ribs (p < 0.1) was observed in newborn
rats exposed by inhalation to 1000 mg/m³ (265 ppm) toluene for 24 hours per day on days 7–14
of gestation (Tatrai et al., 1980).
Fused sternebrae and extra ribs were observed in rats exposed to 400 ppm (1500 mg/m³)
toluene for 24 hours per day on days 9–14 of gestation (Hudak and Ungvary, 1978). Skeletal
retardation was observed in rats exposed to 266 ppm (1000 mg/m³) toluene for 8 hours per day
on days 1–21 of gestation and to 400 ppm (1500 mg/m³) 24 hours per day on days 1–8. This
same group exposed mice to 400 ppm (1500 mg/m³) or to 133 ppm (500 mg/m³) toluene for 24
hours per day on days 6–13 of gestation. All dams died at 400 ppm (1500 mg/m³) and a
statistically significant decrease in fetal weight was observed at 266 ppm (1000 mg/m³).
In another set of experiments, continuous exposure of pregnant rats to higher concentrations of

1000 and 1500 ppm (3800 and 5700 mg/m3) toluene on days 9 to 14 of gestation resulted in the
death of two dams out of 19 during the exposure to 1500 ppm (5700 mg/m3) (Hudak and
Ungvary, 1978). Fetuses from the 1500 ppm (5700 mg/m3) group showed increased incidence
of sternebral alterations, extra ribs and missing tails. The same exposure on days 1 through 8 of
gestation resulted in 5 deaths out of 14 dams. Fetuses exposed to this treatment showed
increased incidence of hydrocephaly and growth retardation compared to controls. A third
treatment that exposed pregnant rats to 1000 ppm (3800 mg/m3) on days 1 through 21 of
gestation resulted in no maternal death, decreased maternal weight gain or fetal loss, but
resulted in an increase in the incidence of skeletal variations in the fetuses.
In Klimisch et al. (1992), skeletal retardations were observed in the offspring of 15 pregnant
rabbits per group exposed by inhalation to concentrations of toluene ranging from 30 to 300
ppm (110 to 1100 mg/m³), 6 hours per day on days 6–18 of gestation, however the frequency of
skeletal retardations was not significant compared with corresponding controls. These results
were not dose-dependent and no effects were seen in the two additional groups of 20 rabbits,
each group exposed to 100 or 500 ppm (380 and 1900 mg/m³) toluene.
A statistically significant increase in the number of animals showing a 13/13 rib profile (which is
considered within the range of normal development) was observed in offspring of female mice
exposed to 400 ppm (1500 mg/m³) toluene, 7 hours per day on days 7–16 of gestation
(Courtney et al., 1986).
Gleich and Hofman (1983) observed an increased number of resorptions in female mice
exposed to 400 ppm (1500 mg/m³) toluene on days 6–15 of gestation; the daily exposure
duration was not specified.
The best available study relating toluene exposure and retardation of somatic development is
one in which adult rats of 2 generations were exposed for 6 hours per day to 0, 100, 500 or
2000 ppm (0, 380, 1900, or 7500 mg/m³) toluene during an 80-day premating period and a 15
day mating period (API, 1985). Adult females of both generations were also exposed on days 1–
20 of gestation and on days 5–21 of lactation. The mean body weights of fetuses of both
generations of dams exposed to 2000 ppm (7500 mg/m³) were significantly decreased
compared to controls. No maternal toxicity was reported. Exposure at 2000 ppm (7500 mg/m³)
to the male parent did not result in any adverse effects.
After weaning, the F1 pups were exposed 80 times (6 hrs per day, 5 days per week) to the
appropriate exposure level and then randomly mated to members of the same exposure group.
The F1 generation exposed to 2000 ppm (7500 mg/m³) toluene showed significantly decreased
body weight which persisted throughout lactation. No effects were observed on histopathology.
No data were presented for the F2 generation. The NOAEL for fetotoxic effects in this study was
500 ppm (1900 mg/m3).
In a more recent teratogenicity study, Ono et al. (1995) exposed pregnant Sprague-Dawley rats
to 600 or 2000 ppm (2300 or 7500 mg/m3) toluene for 6 h/day from day 7 to day 17 of
pregnancy. The control group inhaled clean air. Maternal exposure to 2000 ppm (7500 mg/m3)
caused significant toxic effects such as body weight suppression in dams and offspring, high
fetal mortality, and embryonic growth retardation. However, no external, internal, or skeletal
anomalies were observed in the fetuses of either treated group. In addition, there were no
differences in the results of pre- and post-weaning behavioral tests of the offspring, including
surfacing righting, position adjusting, space exploration and spatial learning. No changes which
could be related to toluene were apparent in the 600 ppm group. Thus, 600 ppm (2300 mg/m3)
is a NOAEL in this study.
Da Silva et al. (1990) exposed pregnant rats and hamsters to 0 or 800 mg/m3 (210 ppm) toluene
for 6 hours/day on gestation days 14–20 (rats), or days 6–11 (hamsters). Fetuses of exposed
rats demonstrated a significant exposure-related decrease in birth weight compared with
controls. In addition to low birth weight, the number of live pups was significantly lower in the
800 mg/m3 (210 ppm) group. No deficits in any parameter were noted in the hamsters. In this
study, offspring of rats and hamsters exposed to toluene performed worse than controls on two
neurobehavioral tests – spontaneous alternation test for rats and rotating rod test for hamsters;
however, the differences are not statistically significant (i.e., p > 0.05).
Hass et al. (1999) exposed female rats to 0 or 1200 ppm (0 or 4500 mg/m3) toluene for 6 hours
per day from day 7 of pregnancy until day 18 postnatal. Developmental and neurobehavioral
effects in the offspring were investigated using a test battery including assessment of functions
similar to those in the proposed Organization for Economic Cooperation and Development
(OECD) Testing Guidelines for Developmental Neurotoxicity Study (OECD, 2006) (physical
development, reflex development, motor function, motor activity, sensory function, and learning
and memory). The exposure did not cause maternal toxicity or decreased offspring viability.
However, lower birth weight, delayed development of reflexes, and increased motor activity in
the open field were noted in the exposed offspring. The exposed female offspring had poorer
scores on a Morris water maze test (they took longer to locate a hidden platform after platform
relocation) at the age of 3.5 months indicating impaired cognitive function. The difference was
not related to impaired swimming capabilities since swim speeds were similar to control values.
The authors stated that exposure to 1200 ppm (4500 mg/m3) toluene during brain development
caused long-lasting developmental neurotoxicity in rats.
Toluene-based solvents are among the most frequently misused psychoactive substances
during pregnancy, and in both animal models and clinical case reports of toluene exposure, the
primary physiological outcome measure of prenatal inhalant exposure is low birth weight (BW).
To clarify the effect of low BW with prenatal and postnatal toluene exposure, the meta-analysis
by Callan et al. (2016) investigated toluene exposure-induced BW differences in non-primate
mammals by applying a systematic review and meta-analytic techniques to the existing peer-
reviewed animal studies modeling prenatal and postnatal exposure to the inhaled solvent
toluene. Among the 288 studies from literature screen, 24 studies were included in the meta-
analysis with a total of 46 control-to-toluene comparisons differing only in the inhaled
concentration of toluene. The software program DSTAT 1.11 was used for analyzing the data
and conducting the meta-analysis. DSTAT quantification of the data showed a total of 26
different concentrations of toluene were administered through the inhalation route, and were
categorized into the following groups: 0–500 ppm (0–1884 mg/m3), 501–2000 ppm (1888–7537
mg/m3), 2001–5000 ppm (7541–18,842 mg/m3), 5001–7500 ppm (18,846–28,263 mg/m3), and
7500 ppm (28,263 mg/m3) and above. The analysis results indicated that the overall weighted
effect size (a measure of deviance from the null hypothesis) d = -0.39, which means that
prenatal toluene exposure resulted in decreased BW. The 95% confidence interval (- 0.42 to -
0.35) does not include 0, indicating that the effect was significant. External inhaled
concentration, route of administration, day of weighing, and toluene exposure magnitude were
identified as modifiers of this correlation.
Callan et al. (2017) studied the effects of prenatal exposure to relatively high concentrations of
toluene (~ abuse levels) on spatial memory performance in adolescent rats using a spatial
learning and memory task, the Morris Water Maze (MWM). Pregnant Sprague-Dawley rats (N =
56) were exposed to 0, 8000, or 12,000 ppm (0, 30,000 or 45,000 mg/m3) of toluene for 15 min
twice daily from gestation day 8 (GD8) through GD20. Male and female offspring (N= 104) were
observed in the MWM for 5 days beginning on postnatal day (PN) 28 and again on PN44. While
prenatal toluene exposed animals did not differ in initial acquisition in the MWM, rats prenatally
exposed to 12,000 ppm toluene displayed performance deficits (significantly greater distances
from the platform) during a probe trial and in reversal learning on PN44 (p < 0.05). The authors
concluded that prenatal exposure to repeated inhaled high concentrations of toluene can impair
spatial memory function that persists into adolescence.
8. Derivation of Reference Exposure Levels

8.1 Toluene Acute Reference Exposure Level
Study Andersen et al., 1983
Study population 16 male humans, mean age = 24 years
Exposure method Inhalation chamber, 0, 10, 40 and 100 ppm (0, 38,
150, 380 mg/m3 respectively)
Duration 6 hours
Critical effects Impaired reaction time and symptoms of
headache, dizziness, feeling of intoxication,
sensory irritation (eye and nose irritation)
LOAEL 380 mg/m3 (100 ppm)
NOAEL 150 mg/m3 (40 ppm)
Time-adjusted exposure 150 mg/m3 (40 ppm) (no time adjustment for
sensory irritation
LOAEL uncertainty factor 1
Interspecies uncertainty factor 1
Toxicokinetic (UFa-k)
Toxicodynamic (UFa-d)
Intraspecies uncertainty factor
Toxicokinetic (UFh-k) 3
Toxicodynamic (UFh-d) 10
Cumulative uncertainty factor 30
Reference Exposure Level 5000 µg/m3 (5 mg/m3; 1300 ppb;1.3 ppm)
Reference Exposure Levels are based on the most sensitive, relevant health effect reported in
the medical and toxicological literature. Acute Reference Exposure Levels are levels at which
infrequent one-hour exposures are not expected to result in adverse health effects (OEHHA,
2008).
The controlled human exposure study by Andersen et al. (1983) is the key study used for acute
REL derivation. Andersen et al. observed nasal mucus flow, lung function, psychometric
performance, and subjective responses in 16 male humans (mean age = 24 years, age range
21 – 32 years) exposed to toluene concentrations of 10, 40 and 100 ppm (38, 150 and 380
mg/m3) for 6 hours. Exposures to 10 and 40 ppm (38 and 150 mg/m3) toluene were without
subjective irritation effects of strong odor sensation. Statistically significant (p < 0.05) subjective
symptomology included eye and/or nose irritation, headache, and feeling of intoxication among
subjects of 100 ppm (380 mg/m3) toluene exposure. In the psychometric performance tests,
there was a borderline significant correlation (0.05 < p <0.10) for the results on three of the eight
tests for the subjects exposed to 100 ppm (380 mg/m3) toluene. In this study, 40 ppm (150
mg/m3) was recognized as a NOAEL and 100 ppm (380 mg/m3) as a LOAEL to derive an acute
REL for toluene.
In the key study by Andersen et al. (1983), the CNS effects (impaired reaction time and
symptoms of headache, dizziness, feeling of intoxication) and sensory irritation to eyes and
nose were the critical effects for the acute toluene inhalation REL derivation, not odor detection.
Odor detection is mediated through the olfactory nerve, having an accommodation process
which is the gradual decrease in odor perception with continued exposure (Cometto-Muniz and
Abraham, 2016), whereas irritation to eyes and nose is mediated primarily through the
trigeminal nerve (Nielsen and Wolkoff, 2017).
Due to the concentration-dependent nature of chemically-related sensory irritation, no time-

adjusted exposure was applied for extrapolation to a 1-hour exposure. Supporting evidence for
no time-adjusted exposure was observed in the animal study by Oshiro et al. (2011). In this
study, some behavioral effects related to neurotoxicity following acute exposure were better
predicted by the brain concentration of toluene rather than by cumulative inhaled dose (C × t). In
addition, the duration- and concentration-dependent biphasic action of toluene on locomotor
activity could confound the derivation in high acute rodent-exposure situations. Our original
1999 acute REL for toluene was based on the same study by Andersen et al. (1983). With a
time-adjusted concentration of 98 ppm (370 mg/m³), an interspecies uncertainty factor of 1 and
an intraspecies uncertainty factor of 10, the resulting acute REL for toluene was 37,000 μg/m³
(37 mg/m3; 9,800 ppb; 9.8 ppm). In contrast, the new acute, 8-hour and chronic RELs for
toluene described in this document were derived using the methodology described in the
noncancer TSD adopted in December 2008 (OEHHA, 2008). This methodology explicitly
considers possible differential effects on the health of infants, children and other sensitive
subpopulations, in accordance with the mandate of the Children’s Environmental Health
Protection Act (Senate Bill 25). As a result, the previous single intraspecies uncertainty factor
was divided into separate toxicokinetic and toxicodynamic factors, and the combined value
changed from 10 to 30 including a toxicodynamic component of 10 to account for the potentially
greater susceptibility of children to neurotoxic effects. A toxicokinetic component of 3 (√10) was
used in this case to account for the sensory irritation effect which would happen before
metabolism. The point-of-departure was changed from a time-adjusted value of 370 mg/m3 to
the NOAEL value of 150 mg/m3 since the critical effect of sensory irritation is concentration-
dependent and not time-dependent. As described above, the exposure was not time-adjusted
for extrapolation to a 1-hour exposure.
A supporting human exposure study by Echeverria et al. (1989) with a similar study design
provided a LOAEL of 150 ppm (570 mg/m3) and a NOAEL of 75 ppm (280 mg/m3). Echeverria
et al. observed statistically significant decrements in several neurobehavioral tests among a
battery of tests conducted at 150 ppm (570 mg/m3). The results from one test, pattern
recognition latency, were statistically significant at 75 ppm (280 mg/m3). The statistically
significant finding at 75 ppm (280 mg/m3) (ANOVA p values = 0.045 and 0.021 for effect and
trend, respectively) was the only one among the battery of 27 tests within seven psychometric
performance measures. Subjective symptoms of eye irritation and headache increased with
dose but statistical analysis was not provided. A dose-dependent increase (p < 0.001) in the
number of subjects observed sleeping was reported and noted by the authors as the best
evidence for neurological effects from toluene exposure. The evidence by Echeverria et al.
suggests that 75 ppm (280 mg/m3) exposure for 7 hours is near the threshold for the NOAEL.
In the current Hot Spots noncancer REL methodology, when data for compound-specific models
of toxicokinetics are unavailable, an overall intraspecies uncertainty factor (UFH) of 30 rather
than 10 (toxicokinetic component, UFH-k =10; toxicodynamic component, UFH-d = √10) can be
used as a default procedure to protect infants’ and children’s health. An example of this is cases
where differences in metabolism and excretion are key to the toxicological activity. In contrast,
for direct-acting chemicals whose site of action is the point of first contact, a UFH-k of √10 may be
sufficient. Where significant concern for toxicodynamic differences larger than three-fold is
present, a larger UF may be applied, such that the total UFH could be larger than 30 (OEHHA,
2008).
Here in this derivation, where the key effect (sensory irritation) is elicited at the point of first
contact, a UFH-k of √10 is used. An intraspecies uncertainty factor with a toxicodynamic
component (UFH-d) of 10 is applied for the use of human studies with normal adult subjects and
to address the human variation in response to substances with nervous system effects,
including sensitive subpopulations such as children (OEHHA 2008), resulting in an overall UF of
30.
8.2 Toluene 8-hour Reference Exposure Level

Study Zavalic et al. 1998c
Study population 41 adult workers for NOAEL, 32 adult
workers for LOAEL, 83 adult workers for
control
Exposure method Occupational inhalation
Continuity 10 m3/day occupational inhalation rate, 8
hours/day, 5 days/week
Duration 15.60 ± 4.61 years (NOAEL);
19.86 ± 5.61 years (LOAEL)
Critical effects Acquired color vision impairment (
Benchmark concentration (BMCL05) 45.1 mg/m3 (12 ppm)
Time-adjusted exposure 32.3 mg/m3 (8.6 ppm) (12 ppm × 8/8hr × 5/7
days/week)
Subchronic uncertainty factor 1 (exposure > 12% of a 70-year lifetime )
Toxicokinetic (UFh-k) 3.9 (Nong et al. 2006)
Reference Exposure Level 830 μg/m3 (0.83 mg/m3; 220 ppb; 0.22 ppm)
The 8-hour Reference Exposure Level is a concentration at or below which adverse noncancer
health effects would not be anticipated for repeated 8-hour exposures (OEHHA, 2008).
In the Air Toxics Hot Spots Program Technical Support Document (TSD) for the Derivation of
Noncancer RELs adopted in 2008, the use of an 8-hour REL was introduced in order to refine
the risk assessment approach for the large number of industrial facilities that operate and emit
chemicals for 8 hours per day, 5 to 7 days per week and to utilize the advanced features in air-
dispersion modeling. The air dispersion modeling in the Hot Spots Program has traditionally
modeled such emissions as if they were uniformly emitted over 24 hours a day, continuously.
Advances in computer capabilities have made it feasible to model more accurately the ground
level concentrations of these emission scenarios by using meteorology obtained during the time
when the facilities are actually operating (generally daytime). The majority of the highly
populated areas in California have significant diurnal-nocturnal meteorological differences that
can affect the magnitude of the modeled risk and location of receptors (OEHHA, 2008).
The 8-hour REL also is protective of offsite workers and children in schools. The chronic
noncancer health impacts on offsite workers (individuals working at other worksites in areas
impacted by the facility emissions) have been traditionally assessed with the chronic RELs.
Because offsite workers generally work 8 hours and not 24, the eight-hour RELs will ensure a
more accurate assessment of the health impacts of their exposures. The eight-hour RELs will
also be useful for assessing the health impacts of exposure of children in schools, who usually
would not spend more than 8 hours a day in schools. Exposure duration for children and offsite
workers will vary, but an eight-hour exposure duration assumption would be reasonable,
particularly if children and offsite workers are exposed to facility emissions at their school or
place of work and not at their residential locations (OEHHA, 2008).
The study by Zavalic et al. (1998c) was selected as the best available study because it
employed human subjects, used a sensitive endpoint (acquired color vision impairment
(dyschromatopsia)), and two toluene exposure concentrations of 35 and 156 ppm (132 and 587
mg/m3). The use of two exposure levels made it possible to perform a benchmark dose analysis.
A LOAEL of 156 ppm (587 mg/m3) and a NOAEL of 35 ppm (132 mg/m3) were estimated for
acquired color vision impairment (dyschromatopsia) using a sensitive color vision testing
method (i.e., Lanthony D-15 desaturated test).
Acquired color vision impairment (dyschromatopsia), reflects neural alterations in the peripheral
system and can be detected before subjects are aware of functional disability (Grant, 1980). As
indicated by Braun et al. (1989), acquired color vision impairment effects can be observed
earlier than putative neuropsychotoxic effects in workers exposed to organic solvents including
toluene. This conclusion is supported by Gobba et al. (2000), who reviewed more than 50
studies published on color perception in workers exposed to neurotoxic chemicals, and
concluded that color vision impairment from chemical exposure is an early effect that can
generally be detected at low exposure levels if the method adopted for color vision testing is
sensitive enough, such as the Lanthony D-15 desaturated test. With the sensitive and early
detectable effects of acquired color vision impairment, the dataset from Zavalic et al. (1998c)
provided the possibility of a lower and more protective REL value than studies on other
neurological effects.
The primary study by Zavalic et al. (1998c) provided the minimal dichotomous data (two
exposure concentrations and a control group) necessary to run a benchmark concentration
analysis using U.S. EPA BMDS software (USEPA, 2020). The BMC models for dichotomous
data gave acceptable line fits to the data with BMD05 values over a range of 5 to 32 ppm (19 to
121 mg/m3) (Table 4). The probit model was chosen to provide the point of departure for the
REL derivation because it had the lowest Akaike Information Criterion (AIC) value, and the
highest p-value for goodness-of-fit, and generated a BMCL05 (12 ppm (45 mg/m3)) at the lower
end of the range (Figure 2).
Use of a BMCL05 in a REL derivation takes into account some of the inter-individual variability
within a population, generally resulting in a reduction of the standard intraspecies uncertainty
factor. However, a worker population such as that used in the Zavalic study is considered
healthier than the human population as a whole (i.e., healthy worker effect). Thus, to be
adequately protective of vulnerable subpopulations, an intraspecies toxicodynamic (UFh-d) factor
of 10 is used to represent differences within the human population. A factor of 1 was used for
the subchronic uncertainty factor because all the worker subjects have been exposed for more
than 8.4 years (i.e., >12% of estimated 70 year lifetime, OEHHA 2006), which is considered a
chronic human exposure. No adjustment for average experimental exposure duration was
applied for occupational exposures of 8 hr/day since the REL is for an 8-hour daily exposure.
An adult-to-child pharmacokinetic adjustment factor of 3.9 for neonates about 1 month of age
was calculated and represented the largest inter-individual variability between adults and
children/infants (Nong et al., 2006). This was used in place of the default intraspecies
uncertainty factor – toxicokinetic component (UFH-k) for which a PBPK model including
measured inter-individual variability is applied.
An intraspecies uncertainty factor – toxicodynamic component (UFH-d) – of 10 is applied to

account for the greater susceptibility of children to neurotoxic effects.
Table 5. Benchmark dose analysis (USEPA BMDS 3.1.2) of data from Zavalic et al., 1998c.
Model BMC05 BMCL05 p-value for AIC*

ppm ppm fit
(mg/m3) (mg/m3)
Probit 16.37 (62) 11.93 (45) 0.9133 197.02
Logistic 16.78 (63) 12.10 (46) 0.8996 197.02
Quantal Linear 11.01 (41) 6.90 (26) 0.8021 197.07
Multistage (β=2) 11.01 (41) 6.90 (26) 0.8021 197.07
*Akaike Information Criterion
Probit Model with BMR of 5% Extra Risk for the BMD and
0.95 Lower Confidence Limit for the BMDL
1
Data
0.9
BMD
0.8 BMDL
0.7 Estimated Probability

Response at BMD
0.6
Response
0.5
0.4
0.3
0.2
0.1
0
0 20 40 60 80 100 120 140
Dose
Figure 2. Probit model fit to Zavalic et al. (1998c) human dyschromatopsia data.
From the PBPK modeling study of Nong et al. (2006), the inter-individual variability factors for
child age groupings indicate that the area under the venous blood concentration vs. time curve
(AUC) of toluene varied only by a factor of up to 3.9 (for neonate group) even though liver
CYP2E1 content can vary by a factor of 20. Due to the age-related changes in other
physiological parameters, the PK variability is less than expected on the basis of age-related
change in the levels of hepatic CYP2E1. The magnitude of the inter-individual variability factor,
in part, can be explained on the basis of CYP2E1 levels in neonates, children, and adults. The
synthesis pathway of the enzyme CYP2E1 is immature at birth followed by rapid onset and
eventual maturation by 6 months to 1 year (Vieira et al., 1996; Cresteil, 1998; Nakamura et al.,
1998; Tanaka, 1998). Using a more extensive analysis, Johnsrud et al. (2003) observed that
maturation of hepatic CYP2E1 content occurred after 3 months, and expression comparable to
adult levels after 1 year. A sensitivity analysis by Nong et al. (2006) showed that hepatic
metabolism of toluene appears to be limited by enzyme content at birth and its
pharmacokinetics evolve gradually to a hepatic blood flow-limited condition with increasing age.
The most recent PBPK models for toluene developed by Mörk et al. (2014) only recognized a
slight difference between adults and infants in terms of toluene metabolism, i.e., the adult-to-
child pharmacokinetic adjustment factor they developed was close to 1. To be adequately
protective for the infants and children, we applied the value of 3.9 derived by Nong et al. (2006)
as the intraspecies uncertainty factor – toxicokinetic component (UFH-k). Although a toxicokinetic
component of 3.9 represents only the first month after birth, we concluded that the most
sensitive members of the population should still be protected from potential adverse effects in
the development of 8-hour and chronic RELs.
Among the available human studies on long-term neurological effects of toluene, Zavalic et al.
(1998c) is the only study that provided clear data supporting both a NOAEL and LOAEL.
Another study that provided both a NOAEL and a LOAEL is Eller et al. (1999), where a control
group, a low-exposure group and a high-exposure group of workers were examined for the
chronic CNS effects of toluene. However, the time-weighted average level of toluene for the
low-exposure group could only be described as below 20 ppm (75 mg/m3), while that for the
high-exposure group could only be described as exceeding 100 ppm (380 mg/m3). Neither
exposure level was clearly defined. Foo et al. (1990) provided a LOAEL of 88 ppm (331
mg/m3), but its “control group” was actually exposed to a low concentration of toluene, and
therefore cannot be regarded as a true control group providing a NOAEL. Thus, Zavalic et al.
(1998c) was chosen over Eller et al. (1999) and Foo et al. (1990) for OEHHA’s 8-hour and
chronic RELs derivation for toluene. In support of the 8-hour REL, an alternative REL (Table 5)
was derived based on the findings of Foo et al. (1990).
Table 5. Supporting 8-hour REL derivation based on study by Foo et al. (1990)
Supportive Study Foo et al. 1990
Study population 30 female electronic assembly workers for
LOAEL, 30 female workers for LOAEL
Exposure method Occupational Inhalation
hours/day, 5 days/week
2.5 ± 2.7 years (LOAEL)
Critical effects Neurobehavioral deficits in 6 out of 8 tests
Time-adjusted exposure 35.0 mg/m3 (9.3 ppm) (13 ppm × 8/8hr × 5/7
days/week)
Subchronic uncertainty factor √10 (8-12% of life time 70 years)
Toxicokinetic (UFh-k) 3.9 (Nong et al. 2006)
The exposure duration was relatively short in the Foo et al. study, necessitating the use of an
uncertainty factor (√10) for lack of chronic exposure. Without this added uncertainty, the
supporting 8-hour REL would have been essentially identical to the 8-hour REL derived in the
key study by Zavalic et al. (1998c).
8.3 Toluene Chronic Reference Exposure Level

Study Zavalic et al. 1998c
Study population 41 adult workers for NOAEL, 32 adult workers
for LOAEL, 83 adult workers for control
Exposure method Inhalation
days/week
19.86 ± 5.61 years (LOAEL)
Critical effects Acquired color vision impairment
(dyschromatopsia) (Table 2)
Benchmark concentration (BMCL05) 45.1 mg/m3 (12 ppm)
Time-adjusted exposure 16.2 mg/m3 (4.3 ppm) (12 ppm × 10 m3/20 m3
× 5 days/7 days)
Subchronic uncertainty factor 1 (>12% of a 70-year life time exposure)
Toxicokinetic (UFh-k) 3.9 (Nong et al., 2006)
The chronic Reference Exposure Level is a concentration at which adverse noncancer health
effects would not be expected from continuous chronic exposures (see Section 7 in the
Technical Support Document (OEHHA, 2008)).
Both the 8-hr and chronic RELs are based on the study by Zavalic et al. (1998c), with Foo et al.
(1990) as a supporting study. The chronic REL derivation is the same, with the exception that
the time-adjusted exposure is based on a 24 hr/day exposure. Studies have shown that color
vision impairment progresses with increasing cumulative exposure to neurotoxic chemicals

including toluene. However, it is unclear whether the effect is reversible or long-lasting if
exposure is discontinued (Gobba and Cavalleri, 2003). Studies with other solvents indicate that
color vision impairment can be a long-term injury (Gobba and Cavalleri, 2003). The resulting
time-adjusted exposure is 4.3 ppm (16.2 mg/m3). The uncertainty factor application is the same
for both 8-hr and chronic RELs.
US EPA (2005) derived a chronic inhalation Reference Concentration (RfC) of 5 mg/m3 for
toluene based on the arithmetic mean of NOAELs (34 ppm) from four studies that measured
either neuropsychological tests results or color vision loss. This introduced uncertainty in
deriving the point of departure from multiple studies with varied endpoints and varied levels of
response. The same time-adjusted exposure was used by both USEPA and OEHHA. However,
USEPA applied an intraspecies UF = 3 for adult-to-child variability based on the
pharmacokinetic information presented in Pelekis et al. (2001). Another 3-fold UF was applied to
account for additional pharmacodynamic and pharmacokinetic factors not accounted for,
resulting in a total UF = 10.
In contrast, the prior chronic REL for toluene was 300 µg/m3 (70 ppb) (OEHHA, 2000). This
chronic REL was derived from a rat inhalation study by Hillefors-Berglund et al. (1995). Male
Sprague-Dawley rats were exposed to 0, 150, 300, 600 or 1200 mg/m3 (0, 40, 80, 160 or 320
ppm) for 4 weeks, 6 hours/day, 5 days/week, followed by a postexposure period of 29-40 days.
The key effects were decreased brain (subcortical limbic area) weight and altered dopamine
receptor (caudate-putamen) binding.
The study LOAEL and NOAEL were 300 mg/m3 (80 ppm) and 150 mg/m3 (40 ppm),
respectively, and the time-adjusted NOAEL was 26 mg/m3 (7 ppm). A cumulative UF = 100 was
used to derive the chronic REL. This cumulative UF consisted of a subchronic UF = 10,
interspecies UF = 1 and an intraspecies UF = 10 with both human toxicokinetic (UFh-k) and
toxicodynamic (UFh-d) factors = 3. An interspecies UF of 1 (instead of the default of 3 generally
used in 2000) was used because a human occupational study (Foo et al., 1990) had a similar
LOAEL (331 mg/m3; 88 ppm).
The Foo et al., 1990 study was not used to develop a chronic REL for toluene because: 1) there
was no true NOAEL group in the human study; 2) the neurotoxicity endpoints observed were felt
to be more sensitive in the animal study than those observable in human studies; and 3) the
Hillefors-Berglund et al. (1995) study was believed to have better exposure characterization
than the Foo et al., 1990 study.
However, the Zavalic et al. (1998) occupational exposure study reported both LOAEL and
NOAEL concentrations (587 mg/m3 (156 ppm) and 132 mg/m3 (35 ppm), respectively). This
study also had acceptable exposure characterization and sensitive neurotoxicity endpoints.
Since OEHHA prefers to use human data to develop RELs where possible, the Zavalic et al.
(1998) occupational exposure study was chosen to develop the current toluene chronic REL.
8.4 Toluene as a Toxic Air Contaminant Especially Affecting Infants

and Children
Under Health and Safety Code Section 39669.5, OEHHA establishes and maintains a list of
Toxic Air Contaminants (TACs) that may disproportionately impact infants and children. OEHHA
evaluates TACs for addition to this list as Reference Exposure Levels for TACs are developed.
Toluene was identified by CARB as a TAC in accordance with section 39657(b) of the California
Health and Safety Code (Title 17, California Code of Regulations, section 93001) (CCR, 2007).
OEHHA considers substances that cause neurotoxicity to disproportionately impact children

(OEHHA, 2001). It has been demonstrated in this report that toluene is neurotoxic in human and
animal models. Animal neurobehavioral studies have also shown that toluene disproportionally
impacts the young, when exposed either prenatally or postnatally, compared to adults (Bruckner
and Peterson, 1981; Batis et al.,2010; Hass et al., 1999; Hougaard et al., 1999). In addition, the
ability of infants to metabolize and detoxify toluene may not reach adult levels until about one
year of age (Cresteil, 1998; Fanucchi, 2006). Thus, infants may be more susceptible to the toxic
effects of toluene due to slower toluene metabolism during the early years of life.
Finally, neonatal effects from human maternal toluene abuse during pregnancy include
intrauterine growth retardation, premature delivery, congenital malformations, and postnatal
developmental retardation. The fetotoxic effects of toluene demonstrated in controlled animal
studies are comparable to humans who have abused toluene-containing products before or
during pregnancy. Intrauterine developmental retardation is the most clearly established effect
in animals, as evidenced by decreased late fetal weight and retarded skeletal development.
There is also limited evidence in rodents for skeletal and kidney abnormalities, as well as
evidence for effects on postnatal physical and neurobehavioral development (Donald et al.,
1991; Grandjean and Landrigan, 2006).
In view of the widespread exposure to toluene as an industrial solvent, increased susceptibility

to the neurotoxic effects on the developing brain in children, the possible reduction in the
metabolism of toluene in the first years of life, and fetotoxicity in newborns resulting from
toluene abuse by pregnant mothers, there is valid concern that toluene exposure may
disproportionately impact infants and children. OEHHA recommends that toluene be identified
as a toxic air contaminant which may disproportionately impact children pursuant to Health and
Safety Code, Section 39669.5(c).
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Air Toxics Hot Spots Program: Toluene Reference Exposure Levels

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TSD for Noncancer RELs OFFICE OF ENVIRONMENTAL HEALTH HAZARD ASSESSMENT

Air Toxics Hot Spots Program

Air and Site Assessment and Climate Indicator Branch

Office of Environmental Health Hazard Assessment

Page Intentionally Left Blank

Office of Environmental Health Hazard Assessment

Toluene Acute REL

Toluene 8-hour REL

Toluene Chronic REL

2. Physical & Chemical Properties (HSDB (2006) except as noted)

3. Major Uses and Sources

5. Acute Toxicity of Toluene

Camara-Lemarroy et al. (2015) assessed 20 patients admitted to an emergency department due

Controlled Chamber Studies

Rahill et al. (1996) conducted an assessment of physical and neuropsychological performance

Von 8 hr at 50, 100, 3 healthy human beings 100 200

Females: 0 and 100

Andersen 6 hr at 0, 10, 40, 16 young healthy males 40 100

Baelum et 7 hr at 0, 93, and 32 males 39 females age 31-50. * 100

Little et al. 20 min at 15 ppm 20 toluene-sensitive patients (9 male, 11 * 15 (56)

Moderate to severe clinical reactions with

Luderer et 3 hr at 50 ppm 10 male 20 female age 19-45. 50 *

Muttray 28-41 min at 200- 8 toluene-exposed workers, 8 controls. 400 *

Borderline impairment (p = 0.06) of color

Lammers 4 hr at 40 ppm. 11 healthy adult males age 20-50. 40 *

Abbreviation: * not observed.

Acute Toxicity to Infants and Children

Acute Toxicity to Experimental Animals

Other Neurological Effects

Using well-established pattern-elicited visual evoked potentials (VEPs) and a physiologically

Acute exposure to toluene results in neurotoxicity including alterations in visual function. N-

Oshiro et al. 775 or 1225 ppm ± 10 40 LPT-trained rats, age = 7.5 - NA NA

Experiment 2 0 (fresh air only) or 1125 14 of 32 LPT-trained rats (age = NA NA

Abbreviations: Br[tol] – brain toluene concentration; C – concentration; LPT - lever-pressing

Experiment 4 Same as Experiment 3 Animal data from Experiment 3 NA NA

6. Chronic Toxicity of Toluene

Community Epidemiological Studies

Wilson (1943) reported that occupational exposure of workers to concentrations of commercial

A battery of neurobehavioral tests was performed in 30 female workers exposed to toluene

Murata et al. (1993) investigated peripheral neurotoxicity in toluene-exposed workers. Subjects

A group of 49 printing-press workers occupationally exposed to toluene for approximately 21.6

Multiple regression analysis found a statistically significant dose-effect relationship between

functions included vibration thresholds, color discrimination and auditory thresholds.

Color vision impairment

Cavalleri et al. (2000) evaluated color vision impairment in 33 toluene-exposed workers.

Other chronic effects

Table 3. Summary of toluene occupational exposure chronic studies in adult workers

Abbate 40 exposed, 12-14 97, Auditory nervous system * 97

Table 3. Summary of toluene occupational exposure chronic studies in adult workers

Chronic Toxicity to Infants and Children

Chronic Toxicity to Experimental Animals

Dopaminergic and serotoninergic neurotransmission activity was significantly altered in various

To determine the neurobehavioral effects of subchronic exposure to toluene, groups of 160

Other CNS effects

To evaluate the potential modifications of subchronic exposure to inhaled toluene on behavior

Study Exposure duration and Subjects / Effects (endpoints) NOAEL LOAEL

Fujimaki et 0 or 50 ppm for 6 hr/day C3H mice / cellular infiltration in NA 50 (190)

Waniusiow 1750 ppm for 6 hr/day, 5 glutathione-depleted guinea pigs / NA 1750

Other chronic effects

Young / Adult Animal Comparisons

7. Developmental and Reproductive Toxicity

An increased incidence of spontaneous abortions was also reported among occupationally